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 cell‐block preparation using Glucomannan extracted from Amorphophallus konjac

To evaluate a cell‐block preparation using glucomannan, which was extracted from Amorphophallus konjac. Ten specimens were centrifuged at 1,500 rpm for 5 minutes, the supernatant was removed; the remnant after the preparation of smear specimens for routine cytological examination was fixed with 20% formalin. The specimen was recentrifuged at 1,500 rpm for 5 minutes, and the supernatant was removed. The residue was resuspended with 2 ml of eosin solution and 1–5 ml of 80% alcohol, and stirred well. After further centrifugation, the supernatant was removed, and one drop of a glucomannan‐formalin water solution was added gently. After immersion in methanol for 2 hours, glucomannan is solidified and becomes gelatinous. The obtained cell block was placed in the cassette for the preparation of tissue specimens, dehydrated by the routine method, infiltrated with paraffin, and a paraffin‐embedded block was prepared. Thin sections were prepared from the paraffin‐embedded cell block, and hematoxylin–eosin (H&E) stain with immunological stains was performed. H&E stain, periodic acid‐Schiff reaction, Alcian blue, and immunohistochemical stain were clearly demonstrated. We evaluated a new modality of cell‐block preparation using a glucomannan‐formalin water solution. We found that the method was easy to perform and thought it could be useful as an alternative technique for cell‐block preparations. Thus, this novel technique should find wide application in the future. Diagn. Cytopathol. 2010;38:652–656. © 2009 Wiley‐Liss, Inc.     

眼组织的透射电镜制样方法

介绍一种眼组织透射电子显微镜标本制备方法。组织先用４％多聚甲醛－２．５％戊二醛混合固定液预固定,１％四氧化锇固定液后固定,丙酮逐级脱水,延长Ｅｐｏｎ８１２包埋液的浸透时间并包埋,用玻璃刀或钻石刀作超薄切片,醋酸主枸橼酸铅双重染色,Ｈ６００－Ⅳ型透射电子显微镜观察。此方法的优点在于眼组织各层超微结构保存良好,没有损伤及变性,我们对该方法的操作要点作了讨论。     

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.     

Three-dimensional architecture of the intrinsic tongue muscles using a modified alkaline maceration method

The three-dimensional architecture of the intrinsic tongue muscle fibers using the anterior part of the rabbit tongue was studied by scanning electron microscopy with a modified chemical-maceration method. The tongue tissues fixed with 10% formalin solution were treated with 1% OsO4 solution at 5 minutes for hardening of the specimen surface. Subsequently, they were immersed in 6N-NaOH solution for 30 minutes at 60 degrees C for the removal of connective tissues followed by dissection of muscle fibers under a binocular microscope to clarify the structure of the intrinsic tongue muscles. The specimens were treated with tannic acid and OsO4 (conductive staining method; Murakami, 1974), and observed with a SEM. Muscular fiber bundles of the transverse and vertical muscles of the tongue changed their direction at the alignment on the sites where the bundles enter the longitudinal muscles from the innermost surface to form monolayers of muscular bundles extending anteroposteriorly. These muscular bundles formed tunnel-like structures each of which covered a longitudinal muscle bundle. It was considered that these tunnel-like structures support the contraction of the longitudinal muscles as the "muscular sheath".     

Silver enhancement of polymerised diaminobenzidine: increased sensitivity for immunoperoxidase staining

Unambiguous identification of lymphocytes is sometimes difficult because of weak immunostaining of the cell membrane immunoglobulins. A simple method of intensifying the diaminobenzidine (DAB) peroxidase reaction was therefore devised. Paraffin wax sections of formalin fixed tonsils and lymphomas were digested with trypsin and immunostained for kappa and lambda light immunoglobulin chains and CD3 antigen by various peroxidase linked detection systems. After reaction with hydrogen peroxide and DAB the sections were immersed in methenamine silver solution at 60 degrees C for three to seven minutes. The light brown stain on the cell membranes of the mantle zone lymphocytes became dark brown and the stronger stain of the plasma cells became black. Mantle zone B lymphocytes and CD3 positive T lymphocytes were precisely outlined even at low magnification and the lymphomas were easily classified as monoclonal or polyclonal. At high magnification, staining was clearer than with the immunogold-silver stain. Cryostat and paraffin wax sections of other tissues immunostained for various antigens showed similar intensification. Silver methenamine provides an easy means of increasing the sensitivity and visual impact of an immunoperoxidase/DAB reaction in any preparation.     

Preparation and use of fixative slides for the diagnosis of Trichomonas vaginalis

Clean microscopic slides are dipped in a solution of mercuric chloride and sucrose in 30% alcohol and allowed to dry. A thin transparent film of cytological fixative is thus formed on both sides of the slide. Fixative slides, prepared in this manner, can be stored indefinitely provided that they are not exposed to a warm, humid atmosphere. When a drop of fluid containing bacteria, protozoa, or mammalian cells is spread over the surface of such a slide the formed elements are immediately fixed, the finer cytological details being well preserved. Staining and examination are carried out when the specimen arrives at the laboratory. This method is particularly suitable for a diagnostic laboratory service in which most of the specimens arrive by mail.     

A reliable method for electron microscopic examination of specific areas from paraffin-embedded tissue mounted on glass slides.

A reliable method for electron microscopic examination of specific areas from paraffin-embedded tissue mounted on glass slides. Am J Clin Pathol 70: 697--699, 1978. A method to embed and cut tissue mounted on ordinary glass histologic preparations for electron microscopic observations is described. The cover slip is removed by immersing the slide in xylene for a few minutes to several hours. After the cover slip is removed, the tissue is fixed in osmium tetroxide, dehydrated, and embedded in epoxy resins by inverting a BEEM capsule filled with the resin on a selected area of the slide. After polymerization, thin sections are obtained and observed with the electron microscope with good results.     

Improving cellularity and quality of liquid‐based cytology slides processed from pancreatobiliary tract brushings

Cytology has been reported to have suboptimal sensitivity for detecting pancreatobiliary tract cancer in biliary tract specimens partly as a result of low specimen cellularity and obscuring noncellular components. The goal of this study was to determine if the use of a glacial acetic acid wash prior to processing would increase the cellularity and improve the quality of ThinPrep® slides when compared to standard non‐gyn ThinPrep processing. Fifty consecutive pancreatobiliary tract specimens containing 20 ml of sample/PreservCyt® were divided equally for standard non‐gyn ThinPrep (STP) and glacial acetic acid ThinPrep processing (GATP). A manual drop preparation was also performed on residual STP specimen to determine the number of cells left in the vial during STP processing. Twenty‐six (52%) specimens had more epithelial cell groupings with the GATP methodology while 19 (38%) had equivalent cellularity with both methods. The STP method produced more epithelial cell groupings in 5 (10%) of the specimens. Of the 26 specimens that had less cells with the STP method, 14 (54%) had ≥50 cell groupings on the manual drop slide processed from the residual STP specimen suggesting that many cells remain in the vial after STP processing. The GATP method was preferred in 25 (50%) of the specimens, the STP method in 5 (10%), while both methodologies provided similar findings in the remaining 20 (40%) of specimens. The data from this study suggests that the GATP method results in more cells being placed on the slide and was preferred over the STP method in a majority of specimens. Diagn. Cytopathol. 2010;38:627–632. © 2009 Wiley‐Liss, Inc.     

Detection of UCH-L1 expression by pre-embedding immunoelectron microscopy with colloidal gold labeling in diseased glomeruli

The purpose of this report was to study the use of pre-embedding immunoelectron microscopy technique with gold and horseradish peroxidase (HRP) labeling in detecting the expression of ubiquitin C-terminal hydrolase 1 (UCH-L1) of podocytes in glomerulonephritis. The specimens of human IgA nephropathy and lupus nephritis were fixed with paraformaldehyde and lysine-HCl buffer, labeled by colloidal gold or HRP, embedded with epoxy resin, and examined under the transmission electron microscope. The high density of gold particles or peroxidase reaction products (DAB) combined with UCH-L1 was obvious in cytoplasm and processes of podocytes. This modified technique of pre-embedding immunoelectron microscopy could perfectly preserve the ultrastructure of kidney and expose antigens which is valuable for clinical diagnostic work and experimental research.     

Assessing the effectiveness of 30% sodium chloride aqueous solution for the preservation of fixed anatomical specimens: a 5‐year follow‐up study

Anatomical specimens used in human or veterinary anatomy laboratories are usually prepared with formaldehyde (a cancerous and teratogenic substance), glycerin (an expensive and viscous fluid), or ethanol (which is flammable). This research aimed to verify the viability of an aqueous 30% sodium chloride solution for preservation of anatomical specimens previously fixed with formaldehyde. Anatomical specimens of ruminant, carnivorous, equine, swine and birds were used. All were previously fixed with an aqueous 20% formaldehyde solution and held for 7 days in a 10% aqueous solution of the same active ingredient. During the first phase of the experiment, small specimens of animal tissue previously fixed in formaldehyde were distributed in vials with different concentrations of formaldehyde, with or without 30% sodium chloride solution, a group containing only 30% sodium chloride, and a control group containing only water. During this phase, no contamination was observed in any specimen containing 30% sodium chloride solution, whether alone or in combination with different concentrations of formaldehyde. In the second phase of the experiment, the 30% sodium chloride solution, found to be optimal in the first phase of the experiment, was tested for its long‐term preservation properties. For a period of 5 years, the preserved specimens were evaluated three times a week for visual contamination, odors, and changes in color and texture. There was no visual contamination or decay found in any specimen. Furthermore, no strange odors, or changes in color or softness were noted. The 30% sodium chloride solution was determined to be effective in the preservation of anatomic specimens previously fixed in formaldehyde.     

Deformation and failure of cartilage in the tensile mode

The aim of this study was to visualize, at the ultrastructural level, the deformation and failure mechanism of cartilage matrix in the tensile mode. Full-thickness dumbbell-shaped specimens were prepared from adult bovines. There were two specimen groups; in the 'parallel' group the specimen axis was parallel to the split lines defining the preferential orientation of the collagen in the articular surface, and in the 'perpendicular' group the specimen axis was perpendicular to the split lines. Specimens were placed with the articular surface uppermost and subjected to a graded series of strain within individual mini-tension devices, while observed with stereomicroscopy and confocal laser scanning microscopy. Thereafter, the changes in the ultrastructure were observed with both scanning and transmission electron microscopy. The mechanism of cartilage failure in the tensile mode comprised the following stages, whether the strain was applied parallel or perpendicular to the split line. (1) At 0% strain a fibrillar meshwork within the articular surface was predominantly orientated in the direction of the split line. (2) As strain increased, the fibrillar meshwork became more orientated in the parallel group and reorientated in the perpendicular group in the direction of the applied strain. (3) After complete reorientation of the fibrillar meshwork in the direction of the applied strain, the initial sign of failure was rupture of the fibrillar meshwork within the articular surface. (4) Subsequently, the rupture rapidly propagated into the deeper layers. Greater strains were required for fibrillar reorientation and complete rupture in the 'perpendicular group' than in the parallel group.     

Rapid techniques for DNA extraction from routinely processed archival tissue for use in PCR.

AIMS--To evaluate the ability of four rapid DNA extraction methods to provide DNA for the polymerase chain reaction (PCR) from routinely fixed, paraffin wax embedded archival tissues. METHODS--Eighteen blocks of various tissues, 18 blocks of cervical cancer specimens, and nine blocks of B cell lymphomas were investigated. Both normal and biopsy specimen sized tissues were studied. DNA was extracted using four methods: boiling for 20 minutes in distilled water; boiling for 20 minutes in 5% Chelex-100 resin solution; 3-hour proteinase K digestion; and 3-hour proteinase K digestion, followed by boiling in 5% Chelex-100. Different exons of the p53 gene, human papillomavirus type 16 (HPV 16) sequence, and immunoglobulin heavy chain (IgH) gene rearrangement were amplified from the extracts. RESULTS--The Chelex boiling, proteinase K digestion, and proteinase K digestion-Chelex boiling methods produced DNA suitable for amplification in all of the 45 samples. Boiling in water yielded insufficient template for the PCR in three of the 45 cases (7%), and in six of 42 positive cases (14%) much fainter bands were observed, mostly when the processed material was either biopsy specimen sized or a B cell lymphoma sample. Fragments of the p53 gene were successfully amplified up to 408 base pairs in water boiled extracts, up to 647 in Chelex boiled preparates, and up to 984 in proteinase K digested and proteinase K digested-Chelex boiled samples, although with decreased sensitivity in the last case. All of the templates were reusable after 3 months of storage at -20 degrees C. CONCLUSIONS--Chelex boiling, proteinase K digestion, and proteinase K digestion followed by Chelex boiling produce suitable templates for the PCR from a large variety of paraffin wax embedded tissues. As the simple 20 minute boiling method in 5% Chelex-100 solution requires minimal manipulation and time, it could be useful, especially in the routine processing of large amounts of material.     

Effect of drawing blood specimens proximal to an in-place but discontinued intravenous solution. Can blood be drawn above the site of a shut-off i.v.?

To assess the effects of drawing blood specimens from a site proximal to an intravenous (IV) infusion line, 24 volunteers were infused with approximately 30 mL of 5% (w/v) dextrose in 0.9% (w/v) NaCl solution. Specimens were drawn proximal to the IV line, while the solution was infusing, and at 1, 2, and 3 minutes after discontinuance of the infusion and assayed for glucose, sodium, chloride, and red blood cell (RBC) count. Control samples were obtained simultaneously from the opposite arm to determine any residual or dilutional effects from the infusing solution. Statistical analysis using the paired sample t-test showed no significant difference of RBCs between arms at or beyond 1 minute. Statistical power analysis reveals that there is a 95% level of certainty that there is less than 1% dilution of the test arm specimen. Analysis of sodium and chloride levels showed no contamination of the test arm specimen at 1 minute, but glucose concentrations still showed an average elevation over control of 5% at 3 minutes. The authors concluded that the drawing of blood specimens proximal to an IV infusion, 3 minutes after its discontinuance, has a clinically negligable dilutional effect but substances present at relatively high levels in the infused solution may still be detected.     

Hypoelectrolytic isoosmotic solution for infusion prevents saline‐induced ultrastuructural artifacts of renal biopsy specimens

Artifacts in the process of specimen preparation are frequent in ultrastructural evaluation of renal biopsy. We hypothesized that the common practice of wrapping kidney biopsy specimens in saline‐soaked gauze to prevent the drying of the specimens could be the major factor of artifacts. In this study, whole kidneys from two male Sprague‐Dawley rats were used. Before fixation, fresh small cubes of kidney tissue were macerated in saline (Saline group) or hypoelectrolytic isoosmotic solution for infusion (HISI group) (Sorita T3 or SOLDEM 3A) for 10 or 30 min. Then, the specimens were processed by 1% OsO₄ in 0.1 M phosphate buffer (pH 7.4) and embedded by EPON 812 for ultramicroscopic analysis. In the Saline group, ultrastructural examination revealed swollen podocyte, swollen capillary protuberance of the mesangium into the glomerular capillary loop, tubular cells with swollen mitochondria and microvilli, and the smooth muscle cells in the arteriolar wall with marked vacuolar degeneration were detected after 10 min maceration in saline and these findings become more pronounced after 30 min maceration. However, in the HISI group, these artifacts were not identified or limited within 30 min. It is postulated that HISI solution could prevent the artifacts, and be used for soaking and wrapping instead of physiologic saline solution.     

Comparison of four-layer radioimmunoassay and electron microscopy for detection of human rotavirus.

A four-layer radioimmunoassay (RIA) using polystyrene beads as the solid phase, anti-rota guinea pig IgG as primary antibody, anti-rota rabbit IgG as secondary antibody, and 125I-labelled sheep anti-rabbit immunoglobulin as indicator antibody has been developed for the detection of human rotavirus in stool specimens. A comparison was made of the developed RIA, routine electron microscopy, and research electron microscopy of 147 unconcentrated stool specimens from patients with infantile gastroenteritis. In routine electron microscopy 17 (11.6%) false-positive or false-negative results were obtained when compared with research electron microscopy. Each specimen positive in research electron microscopy was positive in RIA, and six additional RIA positives were found from 58 electron microscopy negative specimens. A confirmatory test was necessary to find out marginally positive but nonspecific reactions in RIA. The developed radioimmunoassay is slightly more sensitive than research electron microscopy of unconcentrated stool specimens and considerably more sensitive and more specific than routine electron microscopy.     

ABC-immunoperoxidase staining of cytologic preparations: improvement of specificity.

Immunoperoxidase (IP) methods perfected on formalin-fixed, paraffin-embedded tissues (FFPE) and then applied to aspirate smears may result in high background staining and a significant number of false-positive results. This is especially true if polyclonal primary antibodies are used or if aspirates and fluids contain a high interstitial or serum protein content. Because of a recurring problem with antiserum to alpha-fetoprotein (AFP), AFP was selected as the primary test antibody with which to evaluate our avidin-biotin complex (ABC)-IP method. The same method, with diaminobenzidine (DAB) or aminoethylcarbazole (AEC) chromogens, was performed on six types of cytologic preparations of a fresh liver specimen. The liver did not stain for AFP in FFPE and frozen tissue; therefore, it could be used to evaluate potential false-positive staining of direct touch imprints, washed aspirate smears, and cytospins that were both air-dried and alcohol-carbowax-fixed. Initial chromogen incubation times were standardized to give identical results on AFP-positive fixed hepatoma and fetal liver controls. Cytologic preparations immunostained with ABC-IP with AEC chromogen resulted in varying background and hepatocyte staining. In comparison, the ABC-IP method using DAB chromogen resulted in no false-positive results and a clean background. The ABC-IP method with AEC standardized for sensitivity on a fixed tissue control required a markedly shortened chromogen incubation time to preclude significant false-positive staining of cytology specimens. It appears that use of AEC chromogen for this antibody with incubation time standardized on a FFPE tissue control and then applied to cytologic preparations also amplifies nonspecific and background staining, contributing difficulty in assessing a true-positive result.(ABSTRACT TRUNCATED AT 250 WORDS)     

Diagnostic value and cost-effectiveness of on-site evaluation of fine-needle aspiration specimens: review of 5,688 cases

Despite the increasing utilization of the ThinPrep Pap Test (TP), limited data exist regarding the cytomorphologic features and patterns of invasive squamous-cell carcinoma in TP specimens. We analyzed a series of TP specimens from patients with histologically confirmed invasive squamous carcinomas of the cervix. Patients with biopsy-proven invasive squamous-cell carcinoma of the cervix with a TP cervical cytologic specimen within the previous 2 mo were identified. The TP slides were analyzed for overall cellularity (percent circle coverage by epithelial cells), tumor cellularity, tumor diathesis, inflammation, coexistent dysplasia, and keratinization. Tumor cellularity was defined as <5%, 5-50%, and >50% of slide cellularity. In all 13 cases that were identified, a cytologic diagnosis of either invasive squamous-cell carcinoma or suspicious for invasive squamous-cell carcinoma was made. In 7/13 cases (54%), epithelial cells covered <40% of the slide circle. Epithelial cells covered >40% of the slide circle in 6/13 cases (46%). Tumor cellularity covered <5% of the slide circle in 4/13 cases (31%), 5-50% in 7/13 cases (54%), and >50% in 2/13 cases (15%). A tumor diathesis was present in 12/13 cases (92%). Inflammation was absent in 1/13 cases (8%), mild in 8/13 cases (62%), moderate in 2/13 cases (15%), and severe in 1/13 cases (8%). Coexistent dysplasia was identified in 12/13 cases (92%). Keratinization was identified in 9/13 specimens (69%). In the vast majority of patients, a diagnosis of squamous-cell carcinoma was rendered on the TP cervical specimen, despite a pattern of decreased cell coverage. It could be hypothesized that tumor diathesis and inflammation may be the etiology for decreased cellularity by blocking filter coverage by epithelial cells. This cellular pattern with diathesis in the ThinPrep smear may be a useful clue to look carefully for diagnostic cells of squamous-cell carcinoma.     

The ultrastructure of the primate arachnoid granulation, studied with the scanning and transmission electron microscopes

The ultrastructures of the primate arachnoid granulations were observed using the scanning (SEM) and transmission (TEM) electron microscopes. The endothelial cells were slender and overlapped each other. Extracellular spaces which were composed of a network of arachnoid cell processes crisscrossing each other three-dimensionally, were observed under the endothelium. By treating with NaOH (20°C), the collagenous fibers in the specimen were exposed under the SEM. The arachnoid granulations were entirely covered with the fibrous capsule, which was composed of wavy collagenous fibers. Many pores (1–2 μm) were observed on the fibrous capsule. These morphological structures may be closely related to the mechanism for cerebrospinal fluid absorption.