Standard Collection Methods Research Articles

Preanalytical Factors Affecting RIA Measurement of Plasma Kisspeptin

Kisspeptins are peptide products of the KiSS-1 metastasis-suppressor ( KISS1 ) gene and the natural ligands of the G-protein–coupled receptor GPR54. KISS1 was initially investigated as an antimetastasis gene. More recent studies have demonstrated that the kisspeptins are potent stimulators of the hypothalamo-pituitary-gonadal axis. Mice and humans with defective kisspeptin signaling show hypogonadotrophic hypogonadism and impaired sexual development (1)(2). Plasma kisspeptin concentrations are <2 pmol/L in men and nonpregnant women. KiSS-1 mRNA is highly expressed in the placenta, and plasma kisspeptin concentrations increase dramatically, to thousands of picomoles per liter, during pregnancy (3). In addition, plasma kisspeptin is increased in women with gestational trophoblastic tumors, thus raising the possibility of measuring plasma kisspeptin as a novel tumor marker (4). Previous studies that have measured plasma kisspeptin in women during pregnancy have found significantly different concentrations of circulating kisspeptin (3)(4) that may be attributable to differences in preanalytical variables, such as collection tube type, processing times, and storage conditions. Use of a standardized sample collection method …

Sample Handling for Mass Spectrometric Proteomic Investigations of Human Sera

Proteomic investigations of sera are potentially of value for diagnosis, prognosis, choice of therapy, and disease activity assessment by virtue of discovering new biomarkers and biomarker patterns. Much debate focuses on the biological relevance and the need for identification of such biomarkers while less effort has been invested in devising standard procedures for sample preparation and storage in relation to model building based on complex sets of mass spectrometric (MS) data. Thus, development of standardized methods for collection and storage of patient samples together with standards for transportation and handling of samples are needed. This requires knowledge about how sample processing affects MS-based proteome analyses and thereby how nonbiological biased classification errors are avoided. In this study, we characterize the effects of sample handling, including clotting conditions, storage temperature, storage time, and freeze/thaw cycles, on MS-based proteomics of human serum by using principal components analysis, support vector machine learning, and clustering methods based on genetic algorithms as class modeling and prediction methods. Using spiking to artificially create differentiable sample groups, this integrated approach yields data that--even when working with sample groups that differ more than may be expected in biological studies--clearly demonstrate the need for comparable sampling conditions for samples used for modeling and for the samples that are going into the test set group. Also, the study emphasizes the difference between class prediction and class comparison studies as well as the advantages and disadvantages of different modeling methods.

Post-breeding inflammation and endometrial cytology in mares

Endometritis has been reported to be the third most common medical condition of horses. Timely diagnosis and treatment of endometritis in mares increases the chance of pregnancy. Exfoliative endometrial cytology is often used as a clinical tool to evaluate endometrial inflammation through detection of neutrophils. There is a lack of information on the time frame for changes in endometrial cytologic parameters following breeding. The main objectives of this article are to use current information to describe systematic analysis of endometrial cytology using standardized methods for sample collection and interpretation, and discuss how these parameters change in relationship to post-breeding interval and mare susceptibility.

Blood samples collected under venous oxygen pressure from patients with sickle cell disease contain a significant number of a new type of reversibly sickled cells: Constancy of the percentage of sickled cells in individual patients during steady state

We found various levels of a new type of reversibly sickled cell (RSC) with blunt edges in 44 blood samples obtained from 32 steady-state patients with sickle cell disease (SCD) without exposure to air (UnExp-blood). Because these RSCs could be generated in vitro by partial oxygenation of once-deoxygenated SS cells to venous oxygen pressure, we named them "partially oxygenated sickled cells" (POSCs). These RSCs were classified into elongated and non-elongated RSCs, depending on the ratio of the short axis to long axis. The presence of these cells was previously unknown because the standard blood collection method oxygenates most of the POSCs to discocytes due to oxygen in the air space in the needle, syringe, and blood collection tube (Exp-blood). Although the shape of elongated POSCs is similar to that of irreversibly sickled cells (ISCs), POSCs revert to discocytes upon exposure to air. We found the following: (1) the percentage of total sickled cells (total POSCs + ISCs) in UnExp-blood (29.0 +/- 14.5%) was significantly higher than the percentage of sickled cells (mainly ISCs) in Exp-blood (7.3 +/- 5.7%); (2) the percentage of sickled cells in UnExp-blood was specific to individual patients during steady state, while it decreased at the onset of a vaso-occlusive event; and (3) the percentage of sickled cells in UnExp-blood varied widely among steady-state patients (4-56%). This new type of RSC may be used as an internal biomarker to evaluate the disease state of individual patients.

Developing methods and strains for genetic studies in the Saccharomyces bayanus var. uvarum species

For years, Saccharomyces cerevisiae has been used as a model organism to gain insight into complex biological processes. The study of closely related yeast species may be critical for understanding the molecular mechanism of evolution. Among those species, S. bayanus var. uvarum could be particularly pertinent because of the availability of its genome sequence. However, to date, in that species genetic studies are problematical due to the lack of standard strains collection and genetic methods. Here, we have developed heterothallic S. bayanus var. uvarum strains and obtained stable haploid strains. We further used UV-induced mutation and gene disruption to create a collection of auxotrophic derivatives. Finally, we have elaborated or improved methods to cultivate cells, obtain zygotes and spores and to transform this species. All these tools can now be used by the scientific community to study the biology of this species.

The relationship between levels of PCBs and pesticides in human hair and blood: preliminary result.

Human hair as a biologic measure of exposure to persistent organic pollutants (POPs) has some advantages over the more commonly used blood and adipose tissue samples. However, one of the primary limitations is the difficulty in distinguishing between exogenous and endogenous contamination. In addition, there are currently no standardized methods for hair sample collection, washing, and chemical analysis. There is also very limited information describing the correlation between levels of organic contaminants in hair and other body compartments. To explore levels of POPs in blood and hair, samples from 10 volunteers were collected and analyzed for select organochlorine pesticides and 57 individual polychlorinated biphenyl (PCB) congeners. We demonstrated that the method for analyzing organic contaminants in human hair was reliable and reproducible. Washing hair with shampoo decreased levels of PCBs, pesticides, and lipids by 25–33% on average and up to 62% for low-chlorinated congeners. The percentage of lipids and the levels of organochlorines in hair were higher than in serum. We found strong correlation (r = 0.8) between p,p′-DDE (dichlorodiphenyldichloroethylene) levels in hair and blood and moderate correlations for the more persistent PCB congeners, but no correlations or weak correlations for other organochlorines. The present study provides preliminary evidence on the utility of hair analysis for POPs; however, further larger studies are recommended before hair analysis can be successfully applied in epidemiologic studies on POPs.

Assessment of Two Alternative Sample Transport and Fixation Methods in the Microbiological Diagnosis of Bacterial Vaginosis

The standard method for specimen collection and transport for microbiological diagnosis of bacterial vaginosis is an air-dried smear of vaginal secretions, promptly heat- or alcohol-fixed, Gram-stained and scored by Nugent's criteria. TWO ALTERNATIVE METHODS ARE EVALUATED: sending a swab in transport medium to be smeared and Gram-stained in the laboratory two days later; and sending a smear of vaginal secretions sprayed with cytological fixative to the laboratory for Gram staining seven days later. One hundred fifty-two women aged 18 years and older who attended a hospital colposcopy clinic or a community healthy sexuality clinic were studied. This was a prospective study: three vaginal swabs were taken from each patient and handled as described above. Each slide was blindly and independently interpreted by two microbiology technologists. The sensitivity, specificity and coefficient of agreement of the transported swab and cytologically fixed methods were compared with the air-dried smear method. Smears from swabs in transport medium and cytologically fixed smears both had 90% sensitivity and 97% specificity for bacterial vaginosis compared with diagnosis from air-dried smears. Cohen's kappa was 0.88 (95% CI 0.79 to 0.97) for each method. Inter-rater reliability assessed over all slides (all sampling techniques) was excellent (kappa 0.94). For the diagnosis of bacterial vaginosis, both alternative techniques provide results equivalent to air-dried direct smears. A vaginal smear sprayed with cytological fixative provides immediate fixation of material to the slide, permits delays in swab transport and avoids the requirement for transport at a controlled temperature imposed by swabs.

Assessment of two alternative sample transport and fixation methods in the microbiological diagnosis of bacterial vaginosis

BACKGROUND: The standard method for specimen collection and transport for microbiological diagnosis of bacterial vaginosis is an air-dried smear of vaginal secretions, promptly heat- or alcohol-fixed, Gram-stained and scored by Nugent’s criteria. OBJECTIVE: Two alternative methods are evaluated: sending a swab in transport medium to be smeared and Gram-stained in the laboratory two days later; and sending a smear of vaginal secretions sprayed with cytological fixative to the laboratory for Gram staining seven days later. PATIENTS AND METHODS: One hundred fifty-two women aged 18 years and older who attended a hospital colposcopy clinic or a community healthy sexuality clinic were studied. This was a prospective study: three vaginal swabs were taken from each patient and handled as described above. Each slide was blindly and independently interpreted by two microbiology technologists. The sensitivity, specificity and coefficient of agreement of the transported swab and cytologically fixed methods were compared with the air-dried smear method. RESULTS: Smears from swabs in transport medium and cytologically fixed smears both had 90% sensitivity and 97% specificity for bacterial vaginosis compared with diagnosis from air-dried smears. Cohen’s kappa was 0.88 (95% CI 0.79 to 0.97) for each method. Inter-rater reliability assessed over all slides (all sampling techniques) was excellent (kappa 0.94). CONCLUSIONS: For the diagnosis of bacterial vaginosis, both alternative techniques provide results equivalent to air-dried direct smears. A vaginal smear sprayed with cytological fixative provides immediate fixation of material to the slide, permits delays in swab transport and avoids the requirement for transport at a controlled temperature imposed by swabs.

Development of an Argentine Spanish hearing impaired speakers database

Argentine Spanish hearing impaired speech has not been systematically registered or studied. A database with standardized methods for data collection and labeling is presented here. Speech of 600 speakers, adults, and children, with different types of hearing losses is recorded at 44100 Hz and 16 bits. The corpus consists of word lists, grouped by tonality and frequency of use, and five short sentences, containing all the phonemes and allophones. Semi-spontaneous speech, consisting of a description of two sets of simple pictures, is also recorded. Data are acoustically analyzed and labeled at the segmental—graphemic, phonetic and acoustic tiers—and suprasegmental levels, using a labeling method that incorporates psychoacoustic information into a combination of categorical and continuous descriptions of fundamental frequency. Syntactic labels are added to semi-spontaneous speech data. Labeled files are loaded into a relational SQL database to allow different types of queries. A complementary database provides information about personal and medical data, and results of hearing tests. These databases contribute to the description of acoustic-phonetic features of deaf speech, a comparison between speech perception and production, and new approaches for the adjustment of auditory prostheses. [Work supported by FONCYT, CONICET and Mutualidad Argentina de HipoacB̄4usicos.]

Description of a new surveillance system for bloodstream and vascular access infections in outpatient hemodialysis centers.

Bloodstream and vascular access infections are a threat to hemodialysis patients. However, there are few studies of rates of such infections and there are no standardized methods for ongoing data collection. Because of frequent hospitalizations and receipt of antimicrobials, hemodialysis patients are at high risk for infection with drug-resistant bacteria. This article describes a new voluntary national surveillance system. Each month participating dialysis center personnel will record the number of chronic hemodialysis patients that they treat (broken down into four types of vascular access). A one-page form will be completed for each hospitalization or in-unit IV antimicrobial start among these patients. These data will allow calculation, stratified by type of vascular access, of several rates, including hospitalizations, in-unit IV antimicrobial starts, and vascular access infections. For individual dialysis centers, this surveillance system will provide a simple and standardized method for recording data, calculating rates, and comparing rates over time. It is hoped that collection and examination of these data will lead to quality improvement measures. For government and the medical and public health communities, aggregation of these data from many dialysis centers will provide a wealth of information that is not currently available. For further information, or to receive a protocol for this study, contact Elaine R. Miller, RN, MPH, at (404)639-6422 (telephone), (404)639-6459 or 6458 (fax), or erm4@cdc.gov (e-mail:). Information is also available on the CDC website at http:@www.cdc.gov/ncidod/hip/Dialysis/dialysis.+ ++htm.

Measuring environmental tobacco smoke exposure in infants and young children through urine cotinine and memory-based parental reports: empirical findings and discussion

OBJECTIVEThis study examined the reliability and potential biases of two urine collection methods from which cotinine measures were obtained and the validity of memory-based parental reports of their children's exposure...

A simple and reproducible method for collecting nasal secretions in frail elderly adults, for measurement of virus-specific IgA.

The standard method for collection of respiratory secretions, by use of a nasal wash (NW) to measure virus-specific IgA, is problematic in frail elderly adults. Therefore, a simplified collection approach using a nasal swab (NS) is described. NW and NS samples were collected from healthy young and frail elderly adults, and IgA titers to respiratory syncytial virus (RSV) fusion and attachment glycoproteins were determined by enzyme immunoassay. Correlation between IgA titers in NW and NS was excellent for each of the antigens (correlation coefficients,.71-.93). In addition, NS results were reproducible when frail elderly subjects were sampled several weeks apart and were nearly equivalent to results from NW samples. The ability to sample nasal secretions by use of an NS when an NW is not technically feasible will facilitate the study of mucosal immunity to RSV as well as the study of mucosal response to candidate RSV vaccines in frail elderly populations.

Delivered Dialysis Dose with PD Plus Therapy

Aim: To evaluate the effect of PD Plus on weekly Kt/V_urea and creatinine clearance (K_cr) among patients undergoing CAPD/CCPD (continuous ambulatory peritoneal dialysis/continuous cyclic peritoneal dialysis). Methods: The kinetic studies of 92 CAPD and 18 CCPD patients who transferred to PD Plus were analyzed. All patients underwent CAPD/CCPD and PD Plus for a minimum of 3 months. Standard collection methods were used and kinetic indices calculated with the Pack PD Kinetic Modeling program. 57 patients had transport data and were modeled for a target weekly Kt/V_urea ≥2.1 using PD Plus with ≤15 liters dialysate/day. 6 patients were supervised during the collection periods by research nurses. The actual results for all patients (110) and for the supervised patients were compared against the modeled results. Results:45% of the patients achieved a Kt/V_urea ≥2.1 and 47% a K_cr ≥60 liters/1.73 m² with PD Plus, but only 20% did so with CAPD/CCPD. A close correlation between the supervised patients and modeled therapy was observed. Conclusions: Adequate dialysis is possible by using higher fill volumes, the supine position, and optimal dwell times (PD Plus) in most patients. The discrepancy between modeled and achieved dose is likely due to poor compliance with therapy, inadequate training, or poor specimen collection.

The role of outcome studies in allergy and clinical immunology.

Outcomes research is becoming an integral part of the sweeping changes occurring in the U.S. health care system. By developing outcomes monitoring systems in allergic disorders, allergists will be able to collect information showing their commitment to excellent clinical care, which improves the patient's quality of life in a cost-effective way. Outcomes data is also important in determining the number of allergists that need to be trained, appropriate therapeutic regimens for allergic disorders, evaluating differences in practice styles of allergists throughout the country, and in revising practice parameters in allergic disorders. Through the Joint Council of Allergy, Asthma, and Immunology, a subcommittee on outcomes was formed. This subcommittee awarded a national asthma outcomes monitoring project to The Health Institute, New England Medical Center, Boston. This project, which will be in three phases, will generate information on outcomes of board-certified allergists in asthma care. Another goal of this project will be to produce standardized instruments and collection methods, which will be made available for use by all allergists to collect their own outcomes data.

Improving Hospital-Acquired Infection Rates: The CDC Experience

The National Nosocomial Infections Surveillance (NNIS) System, begun in 1970 by the Centers for Disease Control to collect data on hospital-acquired infections, is one of the oldest continuously operating clinical performance indicator systems in the United States. Growth of the system, from 19 to 230 hospitals, has been accompanied by developments such as the evolution from hospitalwide to targeted surveillance, improved data processing and telecommunications for data collection and reporting, and risk adjustment. The NNIS System provides specific, standardized methods for data collection and uses device-associated, device-day rates to risk adjust the data and make it meaningful for interhospital comparison. The system has been used as a tool for improving quality of care through prevention of nosocomial infections. For example, an 800-bed teaching hospital's rate of ventilator-associated nosocomial pneumonia in the surgical intensive care unit-49.5 infections per 1,000 ventilator days-was in excess of the 90th percentile. Improvements in care, including changing tubing and cascades every 48 hours and Ambu bags every 24 hours, as well as increased clinical evaluation of patients, was followed 12 months later by a decrease to 25.8 infections, well below the 90th percentile. Since 1992, staff from NNIS hospitals have met in a biennial conference to learn about advances in nosocomial infection surveillance and to share information with one another on infection control and quality improvement programs. The NNIS experience can be used as a source of guidance for assessing the effectiveness and utility of other indicator systems.

A Survey of Gunshot Residue Analysis Methods

A survey was sent to 80 forensic laboratories in 44 States and two Canadian Provinces concerning methodology in analyzing gunshot residue (GSR) and interpreting the results. Of the 80 surveys, 50 (63%) were returned completed. Questions included standard procedures, collection methods, thresholding problems and specificity of data. These results are compared to a previous survey reported in 1990. Implications for the interpretation and future study of these methods are discussed.

Flow rate measurements in isolated perfused kidney tubules by fluorescence photobleaching recovery

We have developed a new application of the fluorescence photobleaching recovery (FPR) technique for instantaneous measurement of volume flow rates at any axial position along isolated perfused kidney tubules. The method requires fast data acquisition of emitted fluorescence through a photomultiplier (time resolution, 0.5 ms) coupled with differential interference contrast microscopy to measure luminal diameters accurately. While the tubule is perfused in vitro with an impermeant fluorophore (fluorescein sulfonate), a 20-ms bleach pulse reduces the fluorescence in the observation region by 20-25%. Fluorescence recovery is a direct function of perfusate velocity; diffusion plays no significant role in the early phase of recovery. A fluid dynamics approach to data analysis shows that fractional recovery increases linearly with time until t = L/2vm, where L is the length of the observation window and vm is the mean axial velocity. Practically, a linear regression analysis of the early recovery phase allows measurement of vm of up to 0.14 cm/s, i.e., a 40-nl/min flow rate in a 25-microns-diameter tubule. Calibration experiments in small glass tubes perfused at predetermined flow rates demonstrated good accuracy (within 10%) and reproducibility (coefficient of variation, 8.7%). In rat inner medullary collecting ducts microperfused at 4-40 nl/min, the correlation with a standard fluid collection method was excellent (r2 greater than 0.97). The method should also be suitable for the direct measurement of fluid flow rate in kidney tubules or blood vessels microperfused in vivo.

Comparison of a Rapid Bioassessment Method with North Carolina's Qualitative Macroinvertebrate Collection Method

A rapid bioassessment method was compared with the State of North Carolina's standard semi-quantitative collection method. The rapid procedure is essentially a subset of the standard method, but with fewer samples (four) and with collections limited to Ephemeroptera, Plecoptera and Trichoptera (EPT). Collecting and processing time for this EPT method was less than one-half that of the standard collection method, but the more rapid method still produced reliable water quality ratings.

Counterflow centrifugation apheresis for the collection of autologous peripheral blood stem cells from patients with malignancies: A comparison with a standard centrifugation apheresis procedure

Two apheresis methods used to collect hematopoietic stem cells from peripheral blood were compared in eight patients with a variety of malignancies. The standard lymphocyte collection method was alternated with the counterflow centrifugation or lymphocyte surge protocol. The number of clonogenic cells (CFU-GM and BFU-E), the red cell volume, and the number of mononuclear cells in each collection were assessed as well as the changes in circulating leukocytes, platelets, and blood hemoglobin produced by each apheresis procedure. There was no statistically significant difference found in the number of clonogenic cells collected with either method, but the number of mononuclear cells collected with the standard procedure was significantly higher (P = 0.001). The red cell volume collected with the standard procedure was significantly higher, (P = 0.0001), but corrected for the number of mononuclear cells the difference was not significant. The counterflow centrifugation apheresis produced significantly less thrombocytopenia (P = 0.005). The counterflow centrifugation apheresis procedure used collected fewer mononuclear cells than the standard procedure, however, with less red cell contamination but a comparable number of CFU-GM and BFU-E in four hour apheresis procedures. Each collection method resulted in a comparable amount of anaemia and leukopenia but the lymphocyte surge method produced less thrombocytopenia following the collection.

Quantification of Mnemiopsis leidyi (Ctenophora, Lobata) from formalin-preserved plankton samples

Lobate ctenophores were thought to be impossible to quantify using standard plankton collection and preservation techniques. I show that the tentacle bulbs of the lobate species Mnemiopsis leidyi provide a direct measure of numbers and sizes of the ctenophores in formalin-preserved plankton samples. The tentacle bulbs persist intact after preservation in 5 % formaldehyde solution for at least 9 mo. Bulb lengths of Live and preserved specimens were closely correlated with ctenophore wet weights. This method is more accurate and convenient than measures of abundance previously used for M. leidyi, and it may be applicable to other species of ctenophores. Ecological studies of ctenophores, other than a few species of cydippids (i.e. Pleurobrachia spp.), have been hampered because of the difficulty in sampling them quantitatively. Standard plankton collection and preservation methods cause the destruction of lobate ctenophores in the class Tentaculata. This has led to special techniques of collection (Harbison et al. 1978) and preservation (Adams et al. 1976) that are very labor intensive. Lobate ctenophores in the genus Mnemiopsis form conspicuous population blooms during spring and summer in estuaries of the eastern United States. These ctenophores are so abundant that methods have been developed to exclude them from plankton nets and to dissolve them if collected (Heinle 1965 and Burrell & Van Engel 1970, respectively). The most common method of measuring the abundance of Mnemiopsis spp. ctenophores has been to measure their total Live volume from plankton tows (Table 1). Total volumes have been converted to approximate numbers from size measurements of the ctenophores (Table 1). Alternatively, live ctenophores have been counted and measured immediately following plankton tows (Table 1). This method is extremely tedious, and potentially inaccurate for small ctenophores. Le Blanc & Straw (1972) and Kremer & Nixon (1976) used luminescent flashes of the ctenophores to measure abundance. O Inter-Research/Printed in F. R. Germany In the present study, I show that tentacle bulb length increases directly with size (wet weight) in Mnemiopsis leidyi. Metamorphosis from the cydippid larva to the adult form in lobate ctenophores involves disappearance of the tentacle sheaths, reduction of the tentacles, and elongation of the tentacular canals and bulbs (bases) as the tentacles shift orally in position (Hyman 1940). These structures remain after preservation in 5 O/ O formaldehyde solution, and can be used to quantify the abundance and biomass of this lobate ctenophore species. Table 1. Studies in which measures of biomass, size, and abundance of A4nern1opsis spp. ctenophores were made. All data were from live specimens from net tows Volume Length Number (m11 (mm)