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PubMedDate not supplied

Key issues relating to the genetic stability and preservation of cells and cell banks.

The long term maintenance of genetically stable cells is important for ensuring reproducible results and continuity in the advance of microbiology, cell biology and biotechnology. As actively growing cultures, cells are constantly at risk of changing, and the necessity for subculturing living materials increases the chances for genetic change and contamination. Many techniques are available for stabilizing living cells; the method employed must be compatible with the intended use of the culture. The most commonly utilized means of preserving living cells are by freezing to cryogenic temperatures, and freeze-drying. Master stocks are usually maintained at liquid nitrogen or comparable temperatures, while working stocks can be frozen or freeze-dried, and maintained at more economical and easily managed temperatures where possible. However, low temperature techniques may cause damage that can result in genetic change, or potential selection when only a small portion of the population survives. Therefore, a good preservation program must include a comprehensive cell characterization regimen that is applied both before and after preserving the cells to ensure that changes are detected when they do occur. Assurance of long term stability necessitates well designed safekeeping and security measures that include minimizing specimen handling through well designed inventory systems, validation and monitoring of storage temperatures, provisions for backup inventory, and training of personnel. Cell banking also requires good cataloguing and data management practices to avoid duplication and misidentification, and to ensure proper tracking of specimens and ease of access.

Bacteria↗
PubMed1991

Patterns of occupational hand injury in pathology. The interaction of blades, needles, and the dissector's digits.

Details of injuries and exposures to body fluids while handling tissue that were sustained by 36 pathologists (nine residents and 27 staff pathologists) were ascertained by retrospective questionnaire. In the preceding year 56% (20/36) had sustained a cut or needlestick injury. A total of 72 individual injuries occurred and corresponded to one injury for every 2629 surgical specimens handled and for every 37 autopsies performed. Residents reported a five-fold excess in injuries compared with staff pathologists. The lesions described predominantly (67%) involved the oppositional area consisting of the distal thumb, index, and middle fingers of the nondominant hand; during dissection, when opposed to hold or retract tissue, these digits form a surface that is directly exposed to the cutting edge, and injury to this area may be termed dissector's digital injury. Injuries to other areas of the nondominant hand were much less numerous and the dominant hand was rarely injured. Splashes to the face and eyes and glove punctures were frequent. These injury and exposure rates are probably broadly representative of practice in areas of low acquired immunodeficiency syndrome prevalence and may be improved on with increasing concern about human immunodeficiency virus infection. It is likely, however, that development of comfortable protective devices, based on knowledge of the pattern and circumstances of injury, will be necessary to eliminate these occupational hazards.

Fingers↗
PubMed2005

Transbronchial needle aspirates: comparison of two preparation methods.

STUDY OBJECTIVES: Transbronchial needle aspiration has evolved as a key bronchoscopic sampling method. Specimen handling and preparation are underrated yet crucial aspects of the technique. This study was designed to identify which of two widely practiced sample preparation methods has a higher yield. DESIGN: Prospective comparison of two diagnostic methods. SETTING: Tertiary academic hospital. PATIENTS: Consecutive patients undergoing transbronchial needle aspiration. INTERVENTIONS: Transbronchial aspirates were obtained pairwise. One specimen was placed directly onto a slide and smears were prepared on site (ie, the direct technique), and the other specimen was deposited into a vial containing 95% alcohol and further prepared in the laboratory (ie, the fluid technique). In total, 282 pairs of samples were aspirated from 145 target sites (paratracheal, 10 sites; tracheobronchial, 101 sites; hilar, 17 sites; endobronchial or peripheral, 17 sites). MEASUREMENTS AND RESULTS: The measured outcome was the presence of diagnostic material at the final laboratory assessment. At least one diagnostic aspirate was obtained in 66% of 86 investigated patients (small cell lung cancer, 18 patients; non-small cell lung cancer, 47 patients; other diagnoses, 21 patients). The direct technique had a better yield overall than the fluid technique (positive aspirates, 36.2% vs 12.4%, respectively; p < 0.01), as well as after stratification for tumor type and for anatomic site. CONCLUSION: The direct technique is superior to the fluid technique for the preparation of transbronchial needle aspirates.

Adolescent↗
PubMed2005

Complex head and neck specimens and neck dissections. How to handle them.

Dissecting surgical specimens from the upper aerodigestive tract is often difficult because of their complicated anatomy. The local environment dictates the routes of tumour spread and surgical margins at risk, and these features differ for various subsites within this part of the body. The examination of surgical specimens of the upper aerodigestive tract should disclose whether postoperative adjuvant treatment is needed and allow the evaluation of preoperatively performed diagnostic imaging. The aim of this article is to provide a concise guideline for the dissection of specimens from this part of the body.

Dissection↗
PubMed1996

Superficial fine needle aspiration by clinicians: a survey of utilization.

Quality assurance is as much concerned with cost, turnaround time, and use of resources as with test reliability and outcome. The cytopathologist needs to be aware of the knowledge base and expectations of clinicians who perform fine needle aspiration (FNA) biopsies of superficial lesions without technical assistance. To this end, a single correct answer multiple choice questionnaire was sent to 82 procedure-oriented clinical faculty members. Completed answer sheets from 35 faculty members were analyzed. The score for fee and turnaround time was 63.2 per cent and for knowledge of technique 70.6 per cent. The overall score for use of FNA was 64.7 per cent and for specimen handling was 45.6 per cent. Of those 65 per cent of participants who made comments, 30 per cent stressed importance of shorter turnaround time and 27 per cent emphasized the need for training in this area of endeavor. It is concluded that the quality of diagnosis can be improved by placing greater emphasis on training and education, the provision of written material, courses or tutorials in technique, written and verbal feedback regarding specimen adequacy, and by making more extensive use of electronic transmission of results.

Biopsy, Needle↗
PubMed1991

Nutritional considerations in the variability of plasma cholesterol measurements.

The concentration of plasma cholesterol is influenced by biological factors as well as clinical and analytical ones. Day-to-day variation can provide a misleading estimate of plasma cholesterol as it can differ by up to 12%. Seasonal variations occur with elevated concentrations in (northern hemisphere) winter relative to summer, possibly due to diet and life-style influences. Clinical and analytical factors also contribute to the variation. These include the manner with which blood is collected, the nutritional state of the subject and subsequent specimen handling. Analytical factors such as the effectiveness of the reagents and controls chosen, as well as the extent of interference by agents of metabolic or dietary origin, can introduce a bias into the analysis. More control can be exerted over the clinical and analytical factors compared with biological variation.

Blood Specimen Collection↗
PubMed1999

Specimen collection and handling.

Pre-analytical factors should be known and standardized to obtain reliable laboratory test result. If they could not be standardized they should be recognized. The specimen should be representative, properly collected, preserved, and labeled. It is also important to prevent any infection to the patients, the phlebotomists and the environment. The ideal is to get the right sample for the right test at the right time.

Blood Specimen Collection↗
PubMed1979

Enterocolitis due to Yersinia enterocolitica in South Australia.

From August 1976 to July 1977, all faecal specimens (3298) sent to the Enteric Department of the Institute of Medical and Veterinary Science, Adelaide were selectively cultured for Yersinia enterocolitica. Yersinia enterocolitica was isolated from three patients with diarrhoea, one of whom acquired her infection overseas. These organisms were not isolated from faecal or lymph node material collected from a limited number of sheep and pigs found to have enteritis at the time of slaughter. Enteric infection due to Yersinia enterocolitica does not appear to be common in Australia and selective culture methods using cold enrichment techniques do not appear to be justified especially in laboratories handling specimens derived mainly from adults.

Adult↗
PubMed1990

Blood cholesterol concentration: fingerstick plasma vs venous serum sampling.

To assess whether venous and fingerstick blood samples yield similar cholesterol concentrations, we obtained both types of samples simultaneously in 108 volunteers participating in a cholesterol screening program. All samples were analyzed by the same enzymatic method in a standardized laboratory, and pairs of simultaneous samples were measured in the same laboratory run. Cholesterol concentrations in fingerstick-derived plasma were consistently higher than in the venous serum (P less than 0.0001), by a positive bias averaging 3.6%. Cholesterol values in fingerstick plasma also were higher than cholesterol results for venous serum placed in a capillary collection tube (average bias +2.4%). The positive bias of fingerstick plasma vs venous serum results appears to be at least partly due to specimen handling, although a true physiological difference between venous and fingerstick cholesterol concentrations is probably also involved. If a positive bias of this magnitude from fingerstick blood sampling is left unadjusted, substantial numbers of people will be labeled "at risk" and referred to physicians when their true values were actually within the acceptable range.

Blood Specimen Collection↗
PubMed1995

Determination of GI147211 in human blood by HPLC with fluorescence detection.

GI147211 (GG211) is a camptothecin analogue, which exhibits antileukemic and antitumor activity by blocking DNA synthesis. The drug stability considerations and specimen handling were important aspects in method development and validation. This method involves collection of blood at the clinical site, immediate freezing, and storage at -70 degrees C. The lactone form is extracted from blood at physiological pH with a mixture of n-butyl chloride and acetonitrile (4:1); the carboxylate is not extracted under these conditions. After evaporation the extract is injected into an HPLC system with a fluorescence detector set at 378/420 nm. The internal standard used is 6,7-dimethoxy-4-methylcoumarin. The main advantages of the procedure are the separation of lactone and carboxylate by means of extraction, simplified specimen collection at clinical sites and the ability to inject almost all of the extracted material (extraction recovery, 60%) into an HPLC system. The method has been validated over the range 0.15-100 ng ml-1 with sufficient precision and accuracy (coefficient of variation below 10%) to support pharmacokinetic studies. Under the conditions of this procedure, the drug is stable in human blood at -70 degrees C for at least 93 days, as well as through two additional freeze-thaw cycles.

Antineoplastic Agents↗
PubMed1998

Skin biopsy techniques for the internist.

OBJECTIVE: To review three commonly performed skin biopsy procedures: shave, punch, and excision. DATA SOURCES: English-language articles identified through a MEDLINE search (1966-1997) using the MeSH headings skin and biopsy, major dermatology and primary care textbooks, and cross-references. STUDY SELECTION: Articles that reviewed the indications, contraindications, choice of procedure, surgical technique, specimen handling, and wound care. DATA EXTRACTION: Information was manually extracted from all selected articles and texts; emphasis was placed on information relevant to internal medicine physicians who want to learn skin biopsy techniques. DATA SYNTHESIS: Shave biopsies require the least experience and time but are limited to superficial, nonpigmented lesions. Punch biopsies are simple to perform, have few complications, and if small, can heal without suturing. Closing the wound with unbraided nylon on a C-17 needle will enhance the cosmetic result but requires more expertise and time. Elliptical excisions are ideal for removing large or deep lesions, provide abundant material for many studies, and can be curative for a number of conditions, but require the greatest amount of time, expertise, and office resources. Elliptical excisions can be closed with unbraided nylon using a CE-3 or FS-3 needle in thick skin or a P-3 needle on the face. All specimens should be submitted in a labeled container with a brief clinical description and working diagnosis. CONCLUSIONS: Skin biopsies are an essential technique in the management of skin diseases and can enhance the dermatologic care rendered by internists.

Biopsy↗
PubMed1995

Determination of the optimum concentration of decontaminants for the primary isolation of Mycobacterium bovis.

The majority of tissue specimens submitted for the isolation of Mycobacterium bovis contain contaminating microorganisms and therefore require selective decontamination before bacteriological examination. The purpose of this study was to identify the preferred decontaminant amongst four commonly used reagents. The four decontaminants used in the study were 1-hexadecylpyridinium chloride, sodium hydroxide, benzalkonium chloride and oxalic acid. A comparison was made of the toxicity of the four decontaminants for M. bovis and their ability to control contamination. Used at the recommended concentrations, all reagents showed a significant degree of toxicity. The toxicity of the decontaminants for three field strains and one laboratory strain of M. bovis were similar, but a second laboratory strain, AN5, was more susceptible. It was also observed that as the concentration of each reagent decreased an abrupt change from control to lack of control of contaminating micro-organisms occurred. Hexadecylpyridinium chloride was found to be the best all-round reagent because at concentrations that effectively controlled contamination this reagent was the least toxic to M. bovis. A protocol for handling specimens based on an assessment of the risk of contamination is recommended.

Journal Article↗
PubMed1998

Comparison of a portable capillary whole blood coagulation monitor and standard laboratory methods for determining international normalized ratio.

The international normalized ratio (INR) is the current standard for monitoring anticoagulation therapy. Although simple to determine, it normally requires venipuncture and extensive laboratory resources for specimen handling and analysis. The portable capillary whole blood coagulation monitor is an alternative to laboratory venipuncture. Its promoted advantages are: it obtains a blood sample by finger-stick versus venipuncture; rapid turnaround time for results; resultant dosage adjustments (as appropriate) performed in minutes versus hours or days after testing; relative ease of use by nonlaboratory personnel; and potential for home monitoring. This project compared the results of INRs obtained through the venipuncture/laboratory process to INRs obtained by the portable monitoring process at the National Naval Medical Center. A correlation coefficient of 0.97 was determined. The difference in the mean INR results of the two testing methods was not clinically significant (p = 0.269). The portable monitor was determined to be a viable alternative to laboratory testing.

Adult↗
PubMed2002

Studies on the characterisation of the cause of leucoreduction failures, with particular reference to extra gatal events.

The causes of leucodepletion failure are multifactoral and can be related to haematological variability in blood donors or donation, defective filters, poor specimen handling or ageing, and/or the presence of non-adhering leucocyte/platelets. Since refiltering removes all types of leucocytes, including the populations appearing as extra gated events, we have developed a practical method for refiltering the failed leucodepleted components on standard filters and back-flushing the second filter to assess the nature of the WBC sub-population. In practice, recovered leucocytes from red cell filters and whole blood mainly consist of neutrophils. Those from platelet and plasma filters were mainly lymphocyte with considerable differences depending on the type of leucodepletion process. Atypical leucocytes are often seen in some pre-/post-cellular leucofiltered components. These appear characteristically as small WBC with a lower affinity for filter matrix, or as cell fragment, pinched leucocyte or apoptotic cells. Different reagents in use show variable sensitivity in identifying these extra gatal events. Storage of leucodepleted samples also induces different types of abnormality in leucocyte dot plot. A useful practical approach for characterisation of the nature of leucocyte sub-populations causing failure in leucodepleted components is provided.

Blood Cell Count↗
PubMed2002

Laboratory diagnosis of Bartonella infections.

Bartonella species are pathogens of emerging and reemerging significance, causing a wide array of clinical syndromes. In North America and Europe, they are increasingly recognized as a cause of culture negative endocarditis, neuroretinitis, and disease among homeless, HIV-infected, and other immunosuppressed individuals. In South America, bartonellosis continues to plague those in endemic regions and poses a significant threat to travelers in these areas. As the clinician is increasingly faced with these illnesses, which may be difficult to diagnose, laboratory techniques to confirm or refute the diagnosis are becoming increasingly important. Culture methods have improved over the past decade demonstrating increased sensitivity, but still require prolonged periods before isolation of the organism. Specimen handling, media selection, and growth conditions all may affect results and must be optimized in order to provide the highest likelihood of recovering the organism. Pure culture of the bacteria not only provides morphologic information, but also provides material for further diagnostic testing. Work with liquid media, which may provide a more rapid means of cultivation has shown some promise and should continue to be pursued. Improved blood culture techniques were a primary factor in the discovery of Bartonella endocarditis and continued improvements will likely demonstrate further clinical insights. Serologic testing for B henselae infections has become the cornerstone of clinical diagnosis, replacing the skin test that was poorly standardized and posed a potential risk to the patient. Immunofluorescence assays have been well characterized and validated in clinical trials, however they are not universally available. Vero cell cocultivated antigens appear to provide higher sensitivity and specificity when compared with agar-derived antigens. IFA assays are inherently difficult to perform, requiring significant expertise to provide reproducible results. On the contrary, enzyme immunoassays offer ease of use and a high level of reproducibility, however ideal antigens for use in the diagnosis of Bartonella infections have not been clearly identified. Continued work to define antigenic targets of the human response to infection and incorporation of these into a widely available EIA will provide a cost-effective tool for the clinician and epidemiologist alike. Due to the close phylogenetic relationship of B henselae and B quintana, differentiation between these species by serologic means may prove difficult. Molecular techniques including PCR offer high sensitivity and specificity, rapid availability of information, and the ability to differentiate Bartonella organisms at the highest level. Results of studies to date are promising and as methods are refined it will be important to conduct clinical studies to define the role of these assays. In disseminated Bartonella infections such as bacillary angiomatosis, peliosis, endocarditis, and urban trench fever, PCR currently offers the ability to establish the diagnosis when other tests may be unrevealing. For CSD, this technique should be used as a confirmatory technique when the diagnosis is unclear by other means. PCR analysis of blood specimens offers a minimally invasive approach to diagnosis, but clinical data are scarce and further studies are needed. As DNA microarrays move into the clinical arena, specific hybridization probes may allow improved identification and differentiation of Bartonellae at the molecular level.

Antibodies, Bacterial↗
PubMed1992

Storage conditions of blood samples and primer selection affect the yield of cDNA polymerase chain reaction products of hepatitis C virus.

We have noticed that suboptimal specimen processing and storage conditions may cause false-negative results in the detection of hepatitis C virus (HCV) RNA in plasma or serum. To establish the influence of specimen handling in a serological laboratory on the rate of detection of HCV RNA by the cDNA polymerase chain reaction (cDNA-PCR), we tested routine serum samples and fresh-frozen plasma samples from the same bleeding from confirmed anti-HCV-positive blood donors. When primers from the NS3/NS4 region were used, HCV RNA was detected in fresh-frozen plasma from 67% of the donors, whereas positive results were obtained with only 50% of the serum samples that had been subjected to routine serological procedures. Analysis of the same samples with primers from the highly conserved 5'-terminal region (5'-TR) revealed an HCV RNA detection rate of 92% for both the routine and the fresh-frozen samples. However, the yield of the amplification product in routine samples was strongly reduced compared with that in fresh-frozen plasma. Comparison of both primer sets for cDNA-PCR showed that the 5'-TR primer set was 10- to 100-fold more effective in detecting HCV RNA. We also analyzed the effect of storage of whole EDTA-blood and serum at room temperature and at 4 degrees C on the yield of the amplification product. A rapid decline in detectable HCV RNA of 3 to 4 log units was observed within 14 days when whole blood and serum were stored at room temperature. By contrast, no perceptible reduction in the cDNA-PCR signal was found in freshly prepared serum stored at 4 degrees C.

Base Sequence↗
PubMed1984

Minimal breast cancer. An update.

In 1971, minimal carcinoma of the breast was defined as those carcinomas which were duct carcinoma in situ (DCIS), lobular carcinoma in situ (LCIS) and those invasive carcinomas that measured 5 mm or less in greatest diameter. Since then the concept has been challenged; the definition has been modified; there has been new knowledge about the forms of carcinoma included in the definition with reference to natural history and response to therapy and there has been documented experience with patients who have minimal mammary carcinoma. These all re-enforce the validity of the original concept. The author reviews this information as well as that of some of the patients of the Breast Cancer Detection and Demonstration Projects jointly sponsored by the American Cancer Society and the National Cancer Institute, discusses some problems with specimen handling, and identifies areas that need additional study.

Biopsy↗
PubMed1992

Chromosome analysis in spontaneous pregnancy loss: use of placental villus mesodermal core cell cultures.

Culture of placental villus mesodermal core cells, a method developed for chorionic villus sampling, was used for cytogenetic analysis in 168 spontaneous pregnancy losses. Successful karyotype results were obtained in 72% of cases and 87.5% of cases in which placental tissue was available. Culture of placental villus mesodermal core cells is useful in the evaluation of spontaneous pregnancy loss, particularly when tissues of direct fetal origin are unavailable or unsuitable for culture. Details of culture and specimen handling techniques are provided.

Abortion, Spontaneous↗