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A method for preparing and handling cross-sections from early chick embryos for scanning electron microscopy.

A method is described for processing multiple cross-sections from early chick embryos for scanning electron microscopy. Embryos are cut through the desired regions. Sections are affixed to a coverslip with Duco cement and critical point dried by Freon 13 or liquid CO2. This method provides a reliable means for preparing multiple cross-sections from single embryos and eliminates the need for direct handling of brittle tissues after drying.

Animals↗

A method for the determination of ethylenediamine in workroom air.

An ethylenediamine-air mixture was generated in a dynamic gas mixing apparatus, and three different sampling techniques were tested. The analysis was performed using isotachophoresis. Sampling in an impinger flask containing hydrochloric acid (20 mmol/l) gave a quantitative recovery. Desorption losses were noticed when silica gel adsorption tubes were used. Cellulose filter support pads impregnated with oxalic acid were laborious to prepare, and the recovery was high only when freshly prepared filters were used. The use of impingers was found to be the most satisfactory method, and it was used for air monitoring in two factories handling ethylenediamine.

Absorption↗

Rapid plastic embedding is compatible with colorimetric detection following whole mount in situ hybridization in plant specimens.

In performing in situ hybridizations, nonisotopic nucleic acid labeling coupled with colorimetric detection offers a safer, easier and more rapid alternative to using radioactively labeled nucleic acid probes and microscopic autoradiography. Whole mount in situ hybridization is also advantageous, because many samples can be processed identically and the reduced handling of specimens greatly reduces the risk of exposing tissues to RNase(s). The thickness of whole mount specimens, however, often prevents accurate determination of sites of expression within specific tissues. Although post-hybridization embedding and sectioning is a solution to this problem, the precipitate formed following the common colorimetric detection procedure is soluble in the organic solvents used for dehydration prior to embedding. We have developed a dehydration and embedding procedure that takes advantage of the compatibility of L.R. White resin containing 10% (v/v) polyethylene glycol 400, and heat polymerized. The addition of the plasticizer allows L.R. White embedded tissues to be sectioned at 10 microm providing excellent signal contrast.

Colorimetry↗

Handling and pathology reporting of adrenal gland specimens.

Proper treatment of resection specimens by the urologist and pathologist is critical in accurately reporting tumors and tumor-like nodules of the adrenal gland. Clinically inapparent adrenal masses are discovered, with increasing frequency, in the course of diagnostic testing or treatment for conditions that are not related to adrenal diseases. As a consequence an increasing number of relatively small tumors lacking the characteristic gross features of malignancy is detected. There is a need for identification of pathological findings that can improve the diagnostic accuracy and predict patient outcome. The aim of this paper is to review the handling and pathology reporting of adrenal gland specimens in the light of recent advances in this field of pathology.

Adrenal Glands↗

Use of agar in ophthalmic pathology: a technique to improve the handling and diagnosis of temporal artery biopsies, subfoveal membranes, lens capsules, and other ocular tissues.

DESIGN: Prospective laboratory methodology study. PURPOSE: Certain tissues, by virtue of their shape and extreme thinness or pliability, are difficult to position correctly during routine paraffin embedding to provide the optimal orientation for histopathologic studies. Biopsy specimens from temporal arteries must be sampled at different points along the length of the artery. Other tissues such as subfoveal neovascular membranes and fragments of lens capsule lack the thickness and rigidity to be positioned on edge to yield cross-sectional views. The authors' technique improves the orientation and thereby maximizes the histologic information obtained from such specimens. METHODS AND MATERIALS: From January 1, 1990, to April 30, 1999, the authors studied 500 consecutive temporal artery biopsy specimens and 200 successive subfoveal neovascular membranes. RESULTS: Cutting a 20-mm cylindrical fragment of temporal artery at 1- to 1.5-mm intervals yielded approximately 13 to 20 cross-sections along the length of the artery. When the specimens were positioned together and embedded in agar, the pathologist could easily study multiple cross-sections of the artery. Additionally, using the agar technique, the authors were able to obtain cross-sections of other specimens submitted, such as subfoveal neovascular membranes, and studied each of the different layers to evaluate the disease process. By the same method, the authors placed small fragments of lens capsule with underlying cortex on edge and readily identified short, gram-positive coccobacilli consistent with Propionibacterium acnes endophthalmitis. CONCLUSION: The agar technique can greatly improve the quality of diagnostic information gleaned from temporal artery biopsy specimens and other small tissue samples.

Agar↗

Changes in sample collection and analytical techniques and effects on retrospective comparability of low-level concentrations of trace elements in ground water.

Ground-water sampling techniques were modified to reduce random low-level contamination during collection of filtered water samples for determination of trace-element concentrations. The modified sampling techniques were first used in New Jersey by the US Geological Survey in 1994 along with inductively coupled plasma-mass spectrometry (ICP-MS) analysis to determine the concentrations of 18 trace elements at the one microgram-per-liter (microg/L) level in the oxic water of the unconfined sand and gravel Kirkwood-Cohansey aquifer system. The revised technique tested included a combination of the following: collection of samples (1) with flow rates of about 2 L per minute, (2) through acid-washed single-use disposable tubing and (3) a single-use disposable 0.45-microm pore size capsule filter, (4) contained within portable glove boxes, (5) in a dedicated clean sampling van, (6) only after turbidity stabilized at values less than 2 nephelometric turbidity units (NTU), when possible. Quality-assurance data, obtained from equipment blanks and split samples, indicated that trace element concentrations, with the exception of iron, chromium, aluminum, and zinc, measured in the samples collected in 1994 were not subject to random contamination at 1 microg/L. Results from samples collected in 1994 were compared to those from samples collected in 1991 from the same 12 PVC-cased observation wells using the available sampling and analytical techniques at that time. Concentrations of copper, lead, manganese and zinc were statistically significantly lower in samples collected in 1994 than in 1991. Sampling techniques used in 1994 likely provided trace-element data that represented concentrations in the aquifer with less bias than data from 1991 when samples were collected without the same degree of attention to sample handling.

Environmental Monitoring↗

Evaluation of exhibits from a murder case using the lead isotope method and scanning electron microscopy.

We have used a combination of lead isotopes and scanning electron microscopy to determine the relationships between different exhibits in a murder case. Samples involved lead projectiles removed from the deceased's head and a pillow, lead-rich scrapings and particles (gunshot residues) from spent cartridges and a silencer, and particles from a pillowcase. The lead projectiles had the same isotopic composition. with the lead being derived from the same dominantly geologically old source(s). The lead smear from the silencer had the same isotopic composition as the projectiles, and the lead was probably from the same source. The particles from the spent cartridges had varying elemental compositions ranging from PbO to PbCuZn +/- Ba with or without Si and are consistent with derivation from the primer. The lead isotopic compositions of the particles from the spent cartridges show some variations, but these are markedly different from those of the projectiles, indicating lead from a mixture of geologically old and geologically young lead. The particles from the pillowcase were extremely small (usually <50 microm size) and showed varying isotopic compositions, some consistent with the gunshot residue from the cartridges. As the exhibits had been handled extensively prior to the present investigation, including some being sent to North America, there is a high likelihood that handling was not done in clean room environments and may have been contaminated. In this instance, as we were concerned about contamination, especially of the pillowcase, we felt contamination negated use of the results for assistance in proving the innocence or guilt of the accused. A combination of high-precision lead isotope measurements with scanning electron microscopy provides a powerful tool for forensic investigations if precautions are taken in handling the exhibits.

Firearms↗

Immunoperoxidase versus immunofluorescence in the assessment of human renal biopsies.

BACKGROUND: For half a century, immunofluorescence (IF) on frozen sections has been the gold standard for immunohistochemical evaluation of renal biopsy specimens. In routine diagnostic immunohistopathologic evaluation, traditional IF has been replaced to a large extent by immunoperoxidase (IP) methods applied to paraffin sections of formaldehyde-fixed tissue. This is caused in part by the practical disadvantages inherent in the IF method, eg, separate tissue specimen and handling, UV microscopy, fading and impermanence of the label-making archiving, and difficult later investigation. Our aim for the present study is to evaluate IP as an alternative to IF in the diagnostic assessment of renal biopsy specimens. METHODS: Proteolytic antigen retrieval, antibodies effective on deparaffinized sections, a sensitive detection system (Dako EnVision HRP; Dako, Copenhagen, Denmark), and a standardized and rigorously controlled procedure were applied to a series of renal biopsy specimens (n = 81) previously classified by means of light microscopy (LM) and IF. Staining for immunoglobulin G (IgG), IgA, IgM, C1q, and C3c were recorded as positive or negative for IF and IP in paired proportions, presuming that IF was the test standard. RESULTS: Concordant observations were 71% for all (282 of 398 observations), 82% for IgG (65 of 79 observations), and 89% for IgA (72 of 81 observations). The majority of discordant observations (74 of 116 observations) were positive by means of IP, with mesangial deposits of IgM and C1q that were not found by IF. Statistically, there was no significant difference in outcomes between IF and IP for IgG, IgA, and C3c ( P > 0.2). In addition, IP staining allowed simultaneous evaluation of tissue by LM and therefore correlation between tissue structure and immune deposits not readily attained by IF. CONCLUSION: In the present study, it is documented that for the detection of IgG, IgA, and C3c, IP applied to protease-digested deparaffinized sections of formaldehyde-fixed renal tissue is, with few exceptions, equal to IF on frozen sections. The EnVision HRP method used here is several times more effective in terms of primary antibody dilution than earlier existing IP methods, and because the avidin-biotin system is not involved, very little nonspecific background staining will occur. Discordant observations (116 of 398 observations; 29%) were in the majority (91 of 116 observations) due to positive IP findings of IgM and C1q, which deserve additional investigation.

Biopsy, Needle↗

Diversity of flaA genotypes among Campylobacter jejuni isolated from six niche-market poultry species at farm and processing.

PCR-restriction fragment length polymorphism of the flagellin (flaA) gene in Campylobacter jejuni was used to determine the relationships of isolates collected at the farm and throughout processing for six niche-market poultry species. This study focused on two specialty chicken products, poussin and free range, and four other specialty products, squab, duck, guinea fowl, and quail. Cloacal and carcass samples were collected from three flocks from each of the six niche species. Three processing plants in California participated in a 2-year investigation. A total of 773 isolates from farm, posttransport, and the processing plants were genotyped, yielding a total of 72 distinct flaA profiles for the six commodities. Genetic diversity of C. jejuni at the farm was greatest for ducks with up to 12 distinct flaA types in two flocks and least for squab 1 flaA type between two farms. For two of the guinea fowl flocks, one free-range flock, two squab flocks, and all three poussin flocks, the flaA types recovered at the prepackage station matched those from the farm. Cross-contamination of poultry carcasses was supported by the observation of flaA types during processing that were not present at the farm level. New C. jejuni strains were detected after transport in ducks, guinea fowl, and free-range chickens. Postpicker, postevisceration, and prewash sampling points in the processing plant yield novel isolates. Duck and free-range chickens were the only species for which strains recovered within the processing plant were also found on the final product. Isolates recovered from squab had 56 to 93% similarity based on the flaA types defined by PCR-restriction fragment length polymorphism profiles. The 26 duck isolates had genetic similarities that ranged from 20 to 90%. Guinea fowl and free-range chickens each had 40 to 65% similarity between isolates. Poussin isolates were 33 to 55% similar to each other, and quail isolates were 46 to 100% similar. Our results continue to emphasize the need to clean processing equipment and posttransport crates in order to decrease cross contamination between flocks. This study also determined that several strains of C. jejuni had unique flaA types that could only be recovered in their host species.

Animals↗