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PubMed1991

A rapid method for the preparation of yeast for immunoelectron microscopy using Lowicryl HM-20.

We describe a fixation and embedding procedure for the yeast Saccharomyces cerevisiae using Lowicryl HM-20 which is rapid, gives excellent fixation, and avoids the low temperature handling normally associated with embedding in this resin. This procedure yields superior structural preservation when compared to the commonly used rapid embedding procedure which employs Lowicryl K4M. We demonstrate that sections prepared using our rapid procedure are suitable for use in immunogold labelling experiments.

Acrylic Resins↗
PubMed2006

Proteomic analysis of amniotic membrane prepared for human transplantation: characterization of proteins and clinical implications.

Amniotic membrane is commonly exploited in several surgical procedures. Despite a freeze preservation period, it is reported to retain wound healing, anti-angiogenic, antiinflammatory and anti-scarring properties; however, little is known about the active protein content. 2-DE analysis of transplant-ready amniotic membrane (TRAM) was performed. The effects of preservation and processing on amnion proteome were investigated, and the major proteins in the TRAM characterized using mass spectrometry and immunoblotting. This identified a spectrum of proteins including thrombospondin, mimecan, BIG-H3, and integrin alpha 6. Preservation compromises cellular viability resulting in selective elution of soluble cellular proteins, leaving behind extracellular matrix-associated and cell structural proteins. A number of key architectural proteins common to the architecture of the ocular surface were demonstrated in AM, which are involved in homeostasis and wound healing. Handling procedures alter the protein composition of amniotic membrane prepared for transplantation. Without standardization, there will be inter-membrane variation, which may compromise the desired therapeutic effect of transplant ready amniotic membrane.

Amnion↗
PubMed2002

Judging survey quality: local variances.

The present study was undertaken to explore possibilities to judge survey quality on basis of a limited and restricted number of a-priori observations. Here, quality is defined as the ratio between survey and local variance (signal-to-noise ratio). The results indicate that the presented surveys do not permit such judgement. Furthermore, the discussion also suggests that the 5-fold local sampling strategies do not merit any sound judgement in about 10% of all cases. The results further imply that surveys will benefit from procedures, controls and approaches in sampling and sample handling, to assess both average, variance and the nature of the distribution of elemental concentrations in local sites. This reasoning is compatible with the idea of the site as a basic homogeneous survey unit, which is implicitly and conceptually underlying any survey performed.

Air Pollutants↗
PubMed1987

The occurrence of pituitary prolactin depletion-transformation in lactating rats: dependence on strain of rats, homogenization conditions and method of assay.

The objectives of this study were to determine whether pre-release transformation (depletion) of pituitary prolactin occurs as the result of suckling to the same extent in several strains of lactating rats, the molecular nature of the transformed hormone, whether the quantity of transformed (depleted) prolactin recovered is dependent upon the type of homogenization buffer used and whether the method of assay influences the extent to which transformed prolactin is detected. During the course of these experiments other factors such as the methods of handling and storing pituitaries and homogenates were also found to influence the amount of prolactin recovered. The results indicated that transformation of prolactin is a very labile event which is affected by many factors. Strain and supplier of rats was critical to the observation of suckling-induced depletion of prolactin, with Spartan- and Holtzman-derived Sprague-Dawley strains exhibiting the most consistent responses. When transformation was observed, it mattered little which buffer was used for homogenization; however, alkaline or acidic buffers extracted more prolactin than did neutral buffers. Triton X-100 also significantly enhanced the efficiency of extraction by neutral buffers. Maintaining pituitaries on dry ice immediately upon removal from the animal increased the amount of prolactin recovered, as did freezing the homogenate for 1-5 weeks before assay. The addition of the protease inhibitor, benzamidine hydrochloride, did not affect the pituitary content of prolactin. Assay of prolactin by polyacrylamide electrophoresis and densitometry yielded more prolactin than either radioimmunoassay or the Nb2 lymphoma bioassay. The molecular nature of pituitary prolactin, extracted at neutral pH, as judged by gel filtration was altered slightly but consistently by suckling, such that large molecular forms increased at the expense of the smallest molecular form. We conclude from these studies that great care must be exercised when attempting to characterize dynamic changes in pituitary prolactin content in lactating rats. Strain and supplier of rats, methods of handling and storing pituitaries, types of buffers used for homogenization and methods of assay all influence the amount of prolactin recovered and can influence the extent to which rapid changes in pituitary prolactin are detected.

Animals↗
PubMed2006

Quality and reliability of routine coagulation testing: can we trust that sample?

Poor standardization of preanalytic variables exerts a strong influence on the reliability of coagulation testing, consuming valuable health care resources and compromising patient outcome. Most uncertainties emerge from patient misidentification and the procedures for specimen collection and handling. Location of unsuitable venous access or problematic phlebotomies may produce spurious activation of the hemostatic system and hemolytic specimens. Prolonged venous stasis is associated with hemoconcentration and spurious variations of most coagulation assays. Additional pitfalls can be introduced by inappropriate phlebotomy tools and small-gauge needles. Inappropriate filling and mixing of the tube, unsuitable procedures for centrifugation and storage of the specimens are additional aspects that need accurate standardization. Besides traditional preanalytic variables affecting routine coagulation testing, thrombin-generation assays require specific criteria to be accurately fulfilled. These aspects include the type of specimen (platelet-poor plasma, platelet-rich plasma or whole blood), blood collection tubes, storage conditions and the presence of residual platelets. Compliance with new international quality assessment programs, which will also involve coagulation laboratories, encompasses the adoption of suitable strategies for reducing undue variability throughout the whole testing process. Such strategies would not entail extraordinary costs and are affordable with a structured outlay of existing resources, educational policies and compliance with reliable guidelines.

Blood Coagulation Tests↗
PubMed2005

Training nonhuman primates to perform behaviors useful in biomedical research.

Data collected from NHPs that are trained to participate voluntarily in husbandry, veterinary, and research procedures are likely to have particular value. The authors present the results of a series of studies that examined the effects of PRT on the performance by chimpanzees of a variety of biomedically relevant behaviors: presenting their perineum for pinworm testing, providing a semen sample, presenting for an s.c. injection, and presenting for an i.m. injection. The overall trends across studies indicate that PRT techniques have significant value in the handling and management of NHPs in many laboratory research settings, including less variability in the data collected and fewer potential confounding variables, which should lead to important refinements in the definition of NHPs as biomedical research models.

Animals↗
PubMed1995

Surface analysis of biomedical devices.

Modern surface analysis methods are highly effective tools in the development and manufacture of biomedical devices. Improvements in the performance of surface analysis techniques and sample handling are shown to have extended their value throughout device development. The application of these techniques and key improvements in their capabilities are discussed in the light of possible future regulatory requirements, which could include auditing of the surface chemistry of devices during manufacture, packaging, and storage.

Equipment and Supplies↗
PubMed2001

Effects of acute handling stress on whitefish Coregonus lavaretus after prolonged exposure to biologically treated and untreated bleached kraft mill effluent.

Exposure of fish to water of impaired quality has been shown to disrupt the function of the hypothalamo-pituitary-interrenal (HPI) axis and alter the interpretation of data from field studies due to the varying effects of handling and delayed sampling on exposed and reference animals. In the present study, juvenile whitefish, Coregonus lavaretus, were exposed for 6 weeks to diluted (4-8%) untreated and biologically treated bleached kraft mill effluent (BKME) and their response to acute handling was investigated. Liver microsomal EROD activity and glycogen phosphorylase (GPase) activity, in addition to gill Na+-K+-ATPase activity, and blood hemoglobin and hematocrit levels were increased in whitefish exposed for 6 weeks to untreated BKME, whereas those exposed to treated BKME exhibited increased blood hemoglobin and red blood cell K+ concentrations. Both handling procedures, exposure to a shallow water (10 cm, 5 min) and to an air challenge (10 s air/10 s water/30 s air/10 s water/10 s air), resulted in acute physiological stress, as recorded after 5-, 60-, and 120-min recovery periods. Following air exposure, the levels of plasma cortisol, blood glucose, hemoglobin, and hematocrit as well as the liver GPase activity were increased, and liver glycogen concentration decreased in control fish. These responses were attenuated in fish exposed to untreated or treated BKME. Plasma estradiol and testosterone levels were not affected by the BKME exposures or by the air challenge. Handling also resulted in attenuated EROD induction in fish exposed to untreated BKME. According to the present findings, the sensitivity of some widely used cellular and physiological variables may be improved by time-dependent standardization when interpreting data obtained following delayed sampling.

Animal Husbandry↗
PubMed2003

The porcine ear skin as a model system for the human integument: influence of storage conditions on basic features of epidermis structure and function--a histological and histochemical study.

Based on careful tissue processing, detailed structural analysis, and histochemical as well as cytophotometrical evaluation of the epidermis, the study presents data with respect to changes of tissue integrity during two storing modes (room temperature and 4 degrees C) and various storage times of the porcine auricle. Structural degeneration was first noted in the barrier region of the epidermis from where such changes spread, independent of storage conditions, from small horizontal necrotic islands and continuously with increasing storage time. The histochemical results corroborated these observations, emphasizing, however, that the lower epidermal layers seemed intact for a longer time period than the upper layers. Cytophotometrical evaluation of histochemical stainings showed, with regard to the enzyme succinate dehydrogenase, that oxidative metabolism was negatively affected in the early stages of storage, whereas epidermal lipids (neutral fats, glycolipids) remained relatively stable, even during storage at room temperature. In conclusion, it was obvious that the barrier region is the most sensitive element of the porcine ear epidermis. Taking into consideration that this part of the epidermis is most important for permeation studies, it seems reasonable to avoid any storage of porcine auricles at room temperature, and to use only auricles that have been stored at 4 degrees C for not more than 4 to 6 hours, immediately after delivery from the slaughter-house. In this way better tissue preservation can be achieved, whereby the use of shinkage-free water-soluble plastic embedding would generally improve the histological control of structural integrity, and the application of an easy to handle enzyme histochemical procedure (e.g. succinate dehydrogenase demonstration) to unfixed fresh-frozen sections would help to control basic aspects of tissue functions. The results are discussed in relation to the use of porcine integument as a model in human dermatological research.

Animals↗
PubMed1992

Microbial contamination of hydrophilic contact lenses. Part II: Quantitation of microbes after patient handling and after aseptic removal from the eye.

We cultured Soflens (polymacon) contact lenses to determine the number of microorganisms present after patient handling and the number present after patient handling and 5 hours of lens wear. Twenty adapted contact lens patients were each dispensed two pairs of new sterile Soflens contact lenses. Both pairs of lenses were handled by the patients during a single experimental session. Prior to handling lenses, patients were instructed to wash their hands with soap and water, rinse with tap water, and dry with a paper towel. One pair (handled-only) was cultured immediately after handling. The second pair (handled-and-worn) was placed on the eyes by the patient, removed aseptically following 5 hours of wear, and then cultured. All 40 handled-only lenses had viable microorganisms associated with them (mean: 653 colony forming units (CFU) per lens). Bacteria were isolated on all of these lenses and fungi were detected on six. The mean bacterial count for the handled-and-worn lenses was 30 CFU/lens; fewer than half exhibited handled-and-worn lenses was 30 CFU/lens; fewer than half exhibited bacteria and no fungi were isolated. These data suggest that patient handling is a highly significant source of microbial contamination of hydrophilic contact lenses. Also, the number of microorganisms on lenses is significantly reduced (P < 0.0001) after the lens has been worn on the eye.

Asepsis↗
PubMed2001

Quantitative assay for group M (subtype A-H) and group O HIV-1 RNA detection in plasma.

A quantitative HIV-1 test is described based on a competitive RT-PCR assay combined with a sandwich hybridization as a detection system. The internal RNA standard (IS) was designed specifically to be competitive during the amplification and during the hybridization step. Sample viral load determination was carried out with one RT-PCR in the presence of 10(3) IS copies. The HIV-1 copy number was calculated by reference to an external standard curve performed on known and increasing amounts of the reference HIV-1 (Ref HIV-1) RNA co-amplified with a constant amount of the IS RNA. The assay had a linear range from 10(1) to 10(6) HIV-1 copies. HIV-1 strains belonging to the different subtypes from group M, but also group O, were all detected. Absolute quantification of purified HIV-1 RNA copies gave identical results as the AMPLICOR HIV-1 Monitor assay. The quantification of patient's samples was evaluated according to different criteria such as dynamic range, sensitivity, efficacy of material recovery, reproducibility and convenience of sample handling. The microplate format of the assay combined with the colorimetric detection provides a convenient tool and fulfills the requirement for routine molecular diagnostic laboratories.

HIV Infections↗
PubMed2002

Evaluation of microbial culture techniques for the isolation of Pythium insidiosum from equine tissues.

The purpose of this study was to evaluate the effects of sample handling, storage, and culture techniques on the isolation of Pythium insidiosum from infected equine tissues. Tissue and kunker samples obtained immediately posteuthanasia from a horse with subcutaneous pythiosis were used to assess the effects of sample type (kunkers vs. tissues), media type (selective vs. nonselective), storage technique, and storage time on P. insidiosum isolation rate. Overall, isolation rates were higher from fresh kunkers (94.6%) and stored kunkers (76.4%) than from fresh tissues (8.3%) or stored tissues (4.6%). Isolation of P. insidiosum also occurred more often on antibiotic-containing media than on nonselective media for both fresh and stored samples. For samples that were stored for 1-3 days prior to culture, P. insidiosum isolation rates were highest for the following techniques: kunkers stored at room temperature and plated on selective media (100%), kunkers stored at 4 C and then plated on either nonselective (91.7%) or selective (95.8%) media, kunkers stored on cold packs and then plated on either nonselective (93.8%) or selective (100%) media, kunkers stored in ampicillin solution and plated on selective media (100%), and kunkers stored in ampicillin/gentocin solution and plated on selective media (87.5%). For samples stored for 4-5 days, P. insidiosum isolation rates were highest for kunkers stored at 4 C and then plated on either nonselective (81.3%) or selective (87.5%) media, kunkers stored in ampicillin solution and then plated on selective media (87.5%), and kunkers stored in ampicillin/gentocin solution and plated on selective media (87.5%). Results of this study suggest that optimal isolation rates of P. insidiosum from infected equine tissues are achieved by culturing fresh kunkers on selective media. For samples that cannot be processed immediately, acceptable handling techniques include storage at room temperature for up to 3 days, refrigeration for up to 5 days, shipping on cold packs, and storage in antibiotic solution, each combined with subsequent inoculation on selective media.

Animals↗
PubMed1999

Value of lumbar puncture in the diagnosis of genetic metabolic encephalopathies.

Diagnostic testing for genetically determined metabolic disease has for many years relied heavily on the use of generalized screening tests that analyze groups of related compounds in easily accessible peripheral fluids such as plasma and urine. Organic acid profiles in urine and amino acid analysis in plasma are two of the most commonly requested tests; these, together with other protocols that examine peripheral fluids, have been and continue to be invaluable tools. There is, however, an emerging realization that many metabolic encephalopathies do not arise secondary to peripheral metabolic changes but rather have their origins within the central nervous system. In these cases, testing of peripheral fluids might be uninformative. This review is designed to examine the role of cerebrospinal fluid analyses in the investigation of infants and children with undefined encephalopathies. The aims are to review the conditions in which measurement of metabolites in cerebrospinal fluid is critical if a diagnosis is to be made, and to emphasize that considerable forethought is often required to ensure correct collection and handling of cerebrospinal fluid. Thus, fidelity of the diagnostic analytic procedures is maintained. This review will help the pediatric neurologist establish practical diagnostic guidelines that in turn will help in the recognition of recently described conditions. Those conditions can, in general, be identified only after specialized cerebrospinal fluid testing.

Biogenic Amines↗
PubMed1988

[Advice as to the safety precautions for handling infectious material in a veterinary practice laboratory].

The number of microbiological investigations with infectious material increases in veterinary practice. Frequently the working space is cramped so that special security measures are imperative. These measures are discussed in detail. Their disregard can result in laboratory infections with zoonotic agents and in unpleasant lawsuits with bad consequences. The German regulations pertaining to security in laboratories are listed. They should be observed in any case.

Animals↗
PubMed2000

From breast to baby: quality assurance for breast milk management.

Breast milk is a body fluid capable of transmitting blood-borne pathogens when ingested. High-risk infants are frequently fed mother's expressed breast milk and may be at risk if they receive the wrong mother's milk. A multidisciplinary team at this 42-bed Level III regional NICU developed a quality assurance program and audit aimed at reducing the risk of feeding expressed breast milk (EBM) to the wrong high-risk infant in the unit. Changes to the old system included modernizing the handling, storing, and distribution of EBM. Elimination of feeding errors was the priority. A specific protocol to minimize EBM feeding errors became part of the quality assurance program and is consistent with the recommendations of the NICU multidisciplinary team.

Breast Feeding↗
PubMed1985

A plastic bag freezing method for preparation of faeces for analysis.

In order to obtain a completely homogeneous mixture of a normal stool it is found necessary only to homogenize the stool samples for 2 min using a paint-can shaker. A method for handling faeces in chemical analyses is based on a freezing procedure in which the homogenized faeces are frozen in a plastic bag separated into different compartments. The frozen faeces can be cut off and weighed for analysis. Compared to the usual method, the plastic bag freezing method is faster and avoids unpleasant smell.

Feces↗
PubMed1993

[Handling of unconventional pathogens].

This article summarizes decontamination problems in handling transmissible spongiform encephalopathies (TSE) in the field of human pathology. The combination of chemical (i.e. 1 M NaOH, 1 h, room temperature) and physical (i.e. autoclaving for 30 min at 131 degrees C) decontamination methods was proven to be suitable for instruments and other materials. Als for the autopsy of suspected TSE patients, safety measures have to be taken. The brain preparation, in particular, represents a considerable risk which must be minimized by appropriate safety measures. Conventional formaldehyde fixation does not decontaminate tissues! Embedding, cutting, deparaffinization and processing or staining have to be done with cautions; members of staff must be aware of the danger. After the handling of suspect materials, the hands should be washed without brushing the skin. In the case of percutaneous inoculation the injury must be decontaminated immediately using iodine- or phenol-containing preparations, 0.5 M NaOH or 1:3000 potassium permanganate.

Autopsy↗
PubMed2002

Sample handling strategies for the determination of biophenols in food and plants.

The analysis of phenols in samples of plant and food origin attracts considerable attention. However, sample handling is often an ignored feature of the analysis. This review highlights the importance of sample extraction in an analysis and the problems that can arise during this step. Many questions remain unanswered and there is a need to more carefully validate extraction efficiencies. Although many new procedures have been developed the use of traditional techniques still dominates.

Food Analysis↗