Specimen Collection Methods Research Articles

HIV Nucleic Acid Amplification Testing Versus Rapid Testing

We conducted a study comparing the OraQuickADVANCE Rapid HIV-1/2 Antibody Test, Uni-Gold Recombigen HIV Test, Determine HIV 1/2 Ag/Ab Combo, EIA, and pooled nucleic acid amplification testing (NAAT). Men who have sex with men rated tests based on specimen collection method and trust in each test. Among 490 subjects, OraQuick performed on oral fluids ranked highest for specimen collection method but lowest on trust; NAAT scored highest on trust. Among a subset of these subjects, 46% would opt for NAAT if choosing one test. Strategies are needed to increase HIV testing that is accurate and consistent with client preferences.

Comparison of two methods in pharmacokinetic blood specimen collection

Objective To find the scientific and safe blood specimen collection method by comparing two methods and to ensure the efficiency and quality of pharmacokinetic blood specimen collection.Methods 248 volunteers participating the clinical pharmacology experiment were randomly divided into control group and experimental group with 124 volunteers in each group.6 118 blood samples were collected in both groups.In the control group,blood was taken by intravenous catheter needle with 5 ml disposable sterile syringes.However,blood was collected with vacuum blood collection method that took blood by disposable venous blood-taking needle at the experimental group.Hemolysis,delay in collection time,insufficient quantities of specimen,contamination and needle injuries were compared in two groups.Results Hemolysis,delay in collection time,insufficient quantity of sample,contamination and needle injuries in the control group (304,92,1 530,765cases) were all significantly higher than the experimental group (25,15,122,21 cases),the differences were statistically significant ( x2 =243.136 2,55.900 0,1 387.346 4,750.324 7,respectively; P ＜ 0.01 ).Conclusions Disposable venous blood-taking needle is safe and highly effective in pharmacokinetic blood specimen collection.It is worthy to be utilized at the Phase Ⅰ clinical trial ward. Key words: Pharmacokinetics; Blood specimen collection; Disposable venous blood-taking needle; Venous indwelling needle

Molecular analysis of volatile metabolites released specifically by staphylococcus aureus and pseudomonas aeruginosa

BackgroundThe routinely used microbiological diagnosis of ventilator associated pneumonia (VAP) is time consuming and often requires invasive methods for collection of human specimens (e.g. bronchoscopy). Therefore, it is of utmost interest to develop a non-invasive method for the early detection of bacterial infection in ventilated patients, preferably allowing the identification of the specific pathogens. The present work is an attempt to identify pathogen-derived volatile biomarkers in breath that can be used for early and non- invasive diagnosis of ventilator associated pneumonia (VAP). For this purpose, in vitro experiments with bacteria most frequently found in VAP patients, i.e. Staphylococcus aureus and Pseudomonas aeruginosa, were performed to investigate the release or consumption of volatile organic compounds (VOCs).ResultsHeadspace samples were collected and preconcentrated on multibed sorption tubes at different time points and subsequently analyzed with gas chromatography mass spectrometry (GC-MS). As many as 32 and 37 volatile metabolites were released by S. aureus and P. aeruginosa, respectively. Distinct differences in the bacteria-specific VOC profiles were found, especially with regard to aldehydes (e.g. acetaldehyde, 3-methylbutanal), which were taken up only by P. aeruginosa but released by S. aureus. Differences in concentration profiles were also found for acids (e.g. isovaleric acid), ketones (e.g. acetoin, 2-nonanone), hydrocarbons (e.g. 2-butene, 1,10-undecadiene), alcohols (e.g. 2-methyl-1-propanol, 2-butanol), esters (e.g. ethyl formate, methyl 2-methylbutyrate), volatile sulfur compounds (VSCs, e.g. dimethylsulfide) and volatile nitrogen compounds (VNCs, e.g. 3-methylpyrrole).Importantly, a significant VOC release was found already 1.5 hours after culture start, corresponding to cell numbers of ~8*106 [CFUs/ml].ConclusionsThe results obtained provide strong evidence that the detection and perhaps even identification of bacteria could be achieved by determination of characteristic volatile metabolites, supporting the clinical use of breath-gas analysis as non-invasive method for early detection of bacterial lung infections.

Abstract 3616: Validation and fitness testing of a quantitative immunoassay for HIF1α in biopsy specimens

Abstract HIF1α is an important marker of hypoxia in human tumors that is altered in a number of cancers, but a reproducible method to determine changes in HIF1α protein expression in human tumor biopsy specimens has not been available. While HIF1α is being used as a pharmacodynamic marker in clinical trials in the NCI (CTEP# 8610, 8856, 8880), an important limitation in measuring the change in HIF1α induced by cancer therapeutic agents is the lability of the protein, in the presence of oxygen, upon collection of the sample. We have devised a method of specimen collection, handling, and extraction that preserves and stabilizes HIF1α levels in tumor biopsies. Degassed collection buffer yielded a significant increase of recovered HIF1α levels compared to the standard method of flash-freezing. The addition of 2-hydroxyglutarate, an inhibitor of prolyl hydroxylase domain enzyme that is involved in the degradation pathway of HIF1α under normoxic conditions, to the collection buffer showed a trend for improved yield of the HIF1α recovery. Sonication and homogenization with ceramic beads were superior to homogenization with metallic beads or tissue grinding. Employing this specimen-handling method allowed validation of a two-site immunoassay for HIF1α quantitation in solid-tissue extracts generated from tumor biopsies. Intra-assay variability was less than 10%, and inter-assay variability was less than 20%. Accuracy, assessed by spike recovery, was 99 +/− 7%. HIF1α readings declined linearly with decreasing sample load over a range of 1 to 10 µg protein per well. Fitness for purpose was demonstrated by quantifying a reduction in HIF1α protein levels following topotecan or indenoisoquinoline NSC 743400 treatment in an A375 xenograft model, followed by assessment of other xenograft models. HIF1α was also demonstrated to be upregulated under low oxygen tension culture conditions in DU145 human prostate cancer cells. Funded by NCI Contract No. HHSN261200800001E. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3616. doi:1538-7445.AM2012-3616

Dried blood spots versus plasma for the quantitation of HIV-1 RNA using a real-Time PCR, m2000rt assay

High costs and stringent requirements for storage and transport of plasma, often prohibit the availability of HIV viral load quantification in resource-limited settings. Dried blood spots (DBS) represent a better method of specimen collection that removes many of these logistical and technical limitations. The present study aimed to assess the performance of the Abbott m2000rt assay for quantitation of HIV-1 RNA in DBS specimens using plasma as a "gold standard" for comparison. One hundred paired DBS and plasma specimens were collected from patients infected with HIV, who were 18 years and older during routine visits to a private tertiary-care clinic in Chennai, India. HIV-1 RNA was extracted manually and then detected using the m2000rt assay. The mean plasma and DBS viral loads were 4.27 (95% CI: 2.65, 5.88) and 4.14 (95% CI: 1.96, 6.32) log copies/mL, respectively. The overall sensitivity of DBS reached 95%; with sensitivities of 62%, 88% and 100% when stratified by viral load ranges of ≤1000, 1000-3000 and >3000 copies/mL, respectively. An over quantitation of the viral load with DBS was observed in pairs with plasma viral load<3000 copies/mL [d=-0.3 log copies/mL (ranging from -0.1 to 0.6 log copies/mL)]. The study showed a strong concordance in RNA levels between plasma and DBS. The use of DBS specimens should be considered for HIV monitoring and for detection of virologic failure in resource-limited settings.

Abstract C3: Validation and fitness testing of a quantitative immunoassay for HIF1α in biopsy specimens.

Abstract HIF1α is an important marker of hypoxia in human tumors that is altered in a number of cancers, but a reproducible method to determine changes in HIF1α protein expression in human tumor biopsy specimens has not been available. HIF1α is being used as a pharmacodynamic marker in a clinical trial of metronomic Topotecan (CTEP# 8610, 9534). However, an important limitation for studying the response of HIF1α to cancer therapeutic agents is the lability of the protein, in the presence of oxygen, upon collection of the sample. We have devised a method of specimen collection, handling and extraction that preserves and stabilizes HIF1α levels in tumor biopsies. Employing this specimen handling method allowed validation of a two-site immunoassay for HIF1α quantitation in solid tissue extracts, such as tumor biopsies. Intra-assay variability was less than 10% and inter-assay variability was less than 20%. Accuracy, assessed by spike recovery, was 100 +/− 10%. HIF1α readings declined linearly with decreasing sample load over a range of 1 to 10 μg protein per well. HIF1α is being used as a pharmacodynamic marker in a clinical trial of metronomic Topotecan, so fitness for purpose was demonstrated by quantifying a reduction in HIF1α protein levels following topotecan treatment of a xenograft model. HIF1α was also demonstrated to be upregulated under low oxygen tension culture conditions in DU145 human prostate cancer cells. The HIF1α immunoassay is currently being transferred to the NCI's National Clinical Target Validation Laboratory for use in support of NCI-sponsored early clinical trials. This Research has been funded with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C3.

A Systematic Review of Endoglin Gene Expression in Preeclampsia

To synthesize scientific literature that addresses the role of endoglin (ENG) gene expression in preeclampsia (PE). A literature search of PubMed and Ovid MEDLINE was conducted using the keywords endoglin, gene, and preeclampsia. Restrictions included English language and humans. Additional articles were identified/selected for evaluation via PubMed e-mail updates (keywords: endoglin and preeclampsia) and review of article reference lists obtained from the search. The initial 14 abstracts retrieved from the literature search were reviewed and 9 studies were selected for evaluation. Review articles and studies not addressing ENG expression (messenger RNA [mRNA] level) in the context of PE were excluded. An additional six articles were selected from PubMed e-mail updates and reference lists. Data related to study objective, design, setting, subject information, phenotype, tissue type, data collection method, statistics, and results were extracted. Regardless of PE definition, ancestral background, gene expression analysis method, tissue type, and time of specimen collection, endoglin appears to play a role in PE development. Moreover, results suggest that a variety of biological mechanisms have the ability to modulate ENG expression in PE, demonstrating the potential complexity associated with endoglin's role in PE. This review article is the first to systematically synthesize evidence related to ENG expression in PE. Findings can be utilized to design future studies that (a) address methodological limitations observed in the reviewed studies and (b) specifically examine why ENG expression levels are altered and address mechanisms explaining how these alterations are involved in PE development.

Cost-Effectiveness Analysis of Diagnostic Options for Pneumocystis Pneumonia (PCP)

BackgroundDiagnosis of Pneumocystis jirovecii pneumonia (PCP) is challenging, particularly in developing countries. Highly sensitive diagnostic methods are costly, while less expensive methods often lack sensitivity or specificity. Cost-effectiveness comparisons of the various diagnostic options have not been presented.Methods and FindingsWe compared cost-effectiveness, as measured by cost per life-years gained and proportion of patients successfully diagnosed and treated, of 33 PCP diagnostic options, involving combinations of specimen collection methods [oral washes, induced and expectorated sputum, and bronchoalveolar lavage (BAL)] and laboratory diagnostic procedures [various staining procedures or polymerase chain reactions (PCR)], or clinical diagnosis with chest x-ray alone. Our analyses were conducted from the perspective of the government payer among ambulatory, HIV-infected patients with symptoms of pneumonia presenting to HIV clinics and hospitals in South Africa. Costing data were obtained from the National Institutes of Communicable Diseases in South Africa. At 50% disease prevalence, diagnostic procedures involving expectorated sputum with any PCR method, or induced sputum with nested or real-time PCR, were all highly cost-effective, successfully treating 77–90% of patients at $26–51 per life-year gained. Procedures using BAL specimens were significantly more expensive without added benefit, successfully treating 68–90% of patients at costs of $189–232 per life-year gained. A relatively cost-effective diagnostic procedure that did not require PCR was Toluidine Blue O staining of induced sputum ($25 per life-year gained, successfully treating 68% of patients). Diagnosis using chest x-rays alone resulted in successful treatment of 77% of patients, though cost-effectiveness was reduced ($109 per life-year gained) compared with several molecular diagnostic options.ConclusionsFor diagnosis of PCP, use of PCR technologies, when combined with less-invasive patient specimens such as expectorated or induced sputum, represent more cost-effective options than any diagnostic procedure using BAL, or chest x-ray alone.

P3-S1.15 The molecular diagnosis of rectal GC and CT infections using the FTA elute card for specimen collection and the real-time multiplex PCR for detection

Background To evaluate the potential use of the Whatman Indicating FTA Elute Micro Card for collection, transport, and storage of rectal swab specimens for subsequent detection of STD pathogens in MSM using a TaqMan-based real-time multiplex PCR. Methods Two rectal swabs were obtained by the physician from each participant in a study to determine carriage of STD pathogens among MSM. One of the swabs was placed into a tube containing 1 ml of Genelock transport medium and while the other swab was firmly pressed onto an FTA Card with three side to side motions held at approximately 60° angle each time. Genomic DNA was eluted from three discs (3 mm diameter) punched out of each card after storage at room temperature for up to 6 months and tested by a real-time multiplex PCR assay which simultaneously detects lymphogranuloma venereum (LGV), non- LGV, Chlamydia trachomatis (CT), and Neisseria gonorrhoeae (GC), and human DNA control. For comparison, an aliquot of sample from genelock tube was extracted using the Qiagen DNA mini kit and tested with same real-time multiplex PCR assay. Results Using purified DNA obtained from the Genelock specimens, the real-time multiplex PCR assay detected nine GC and 17 non-LGV CT; while 7 GC and 15 non-LGV CT were detected in DNA samples eluted from the FTA Cards and used directly for PCR. There were three GC and non-LGV co-infections and no LGV was detected using both specimen collection methods. A substantial number of specimens were found to be PCR inhibitory either collected in Genelock (18.3%) or on FTA Cards (15.4%). Conclusions The FTA Elute Micro Card allows stable storage and convenient transport of rectal swab specimens at room temperature. DNA can be eluted from the card with simple processes instead of numerous purification procedures for downstream real-time PCR amplification and aetiology detection. This preliminary evaluation shows the potential use of FTA Card for rectal specimen collection and PCR testing, and may also provide a cost-saving alternative to expensive international shipping of specimens on dry ice from remote study sites.

Update on bacterial detection methods in chronic rhinosinusitis: implications for clinicians and research scientists

The exact etiologic role of microbes in chronic rhinosinusitis (CRS) remains unclear, due in part to inconsistencies and difficulties of microbiological detection. However, methodological advances now permit comprehensive analysis of microbial communities of the sinonasal region. This review summarizes recent developments in microbial detection technologies as exemplified by their application in the study of CRS. A variety of novel methods for specimen collection, microbial culture, cultivar identification, and microscopic analysis of CRS specimens have been used in recent studies. Moreover, the advent of culture-independent methodologies based on the detection of microbial nucleic acids has greatly expanded the range of microorganisms, including fastidious species, that can be assayed. Many techniques use biochemical interactions to identify nucleic acids as markers for specific species. Technological innovations in microbiology have radically improved the sensitivity and accuracy of microbial detection and identification in CRS specimens. Further application of these microbiological tools to CRS research should provide greater insight into the roles of microbial pathogens and commensals in sinus health and disease. Ultimately, translation of research results into clinical diagnostic technology will improve patient outcomes in this chronic disease.

Validation of two methods for the identification of bacteria directly from blood cultures with the MALDI-TOF technique

Aim We validated two methods for the direct identification of bacteria from positive BACTEC blood culture bottles with the MALDI-TOF technique. Methods 872 blood culture bottles positive with the BACTEC system (BD) were tested. For MALDI-TOF identification two specimen collection and blood cell extraction methods were tested [Method 1: Vacutainer serum seperator tubes (BD); Method 2: MALDI Sepsityper Solutions (Bruker)]. For both methods, bacterial proteins were extracted by the formic acid/ acetonitrile standard method and measured in a MALDI-TOF spectrometer of the FLEX series (Bruker Daltonics). Identification results were compared to routine biochemical methods (VITEK 2, api-system, BioMe´rieux). Results Of 789 blood cultures tested with Method 1, 636 (80%) showed a valid (score value > 1,7) identification result with the MALDI-TOF. Of these valid samples, 630 (99%) were correctly identified to species level in comparison to routine biochemical methods. For Method 2, 83 blood cultures were tested, 75 (80%) were considered as valid, 99% showed a correct result to species level. Discussion The MALDI-TOF technique proved to be a fast and reliable method for the detection of bacteria directly from BACTEC blood culture bottles. Results are available 1–2 days earlier in comparison to conventional methods. We validated two methods for the direct identification of bacteria from positive BACTEC blood culture bottles with the MALDI-TOF technique. 872 blood culture bottles positive with the BACTEC system (BD) were tested. For MALDI-TOF identification two specimen collection and blood cell extraction methods were tested [Method 1: Vacutainer serum seperator tubes (BD); Method 2: MALDI Sepsityper Solutions (Bruker)]. For both methods, bacterial proteins were extracted by the formic acid/ acetonitrile standard method and measured in a MALDI-TOF spectrometer of the FLEX series (Bruker Daltonics). Identification results were compared to routine biochemical methods (VITEK 2, api-system, BioMe´rieux). Of 789 blood cultures tested with Method 1, 636 (80%) showed a valid (score value > 1,7) identification result with the MALDI-TOF. Of these valid samples, 630 (99%) were correctly identified to species level in comparison to routine biochemical methods. For Method 2, 83 blood cultures were tested, 75 (80%) were considered as valid, 99% showed a correct result to species level. The MALDI-TOF technique proved to be a fast and reliable method for the detection of bacteria directly from BACTEC blood culture bottles. Results are available 1–2 days earlier in comparison to conventional methods.

Proposed BioRepository platform solution for the ALS research community

ALS is a rare disorder whose cause and pathogenesis is largely unknown ( 1 ). There is a recognized need to develop biomarkers for ALS to better understand the disease, expedite diagnosis and to facilitate therapy development. Collaboration is essential to obtain a sufficient number of samples to allow statistically meaningful studies. The availability of high quality biological specimens for research purposes requires the development of standardized methods for collection, long-term storage, retrieval and distribution of specimens. The value of biological samples to scientists and clinicians correlates with the completeness and relevance of phenotypical and clinical information associated with the samples ( 2 , 3 ). While developing a secure Web-based system to manage an inventory of multi-site BioRepositories, algorithms were implemented to facilitate ad hoc parametric searches across heterogeneous data sources that contain data from clinical trials and research studies. A flexible schema for a barcode label was introduced to allow association of samples to these data. The ALSBank™ BioRepository platform solution for managing biological samples and associated data is currently deployed by the Northeast ALS Consortium (NEALS). The NEALS Consortium and the Massachusetts General Hospital (MGH) Neurology Clinical Trials Unit (NCTU) support a network of multiple BioBanks, thus allowing researchers to take advantage of a larger specimen collection than they might have at an individual institution. Standard operating procedures are utilized at all collection sites to promote common practices for biological sample integrity, quality control and associated clinical data. Utilizing this platform, we have created one of the largest virtual collections of ALS-related specimens available to investigators studying ALS.

PS1-11: Specimen Collection Within the Cancer Research Network: A Critical Appraisal

Background and Aims: The Genomics Working Group (of the Cancer Research Network (CRN) Pharmacovigilance Project) explored single-site experiences with genomic studies to understand challenges and opportunities for collecting and using biological specimens. Its specific aims were to: describe the availability of existing biospecimens; document successful past specimen collection strategies; identify potential barriers to selected recruitment strategies; and develop standardized study documents for future genomic research. Methods: We distributed a survey on existing biospecimen availability to principal investigators at all CRN Pharmacovigilance sites. Information collected included the number of specimens, storage methods, and type of specimens available. Investigators were also asked to identify past and current studies at their institutions in order to collect information on successful specimen collection strategies. A literature review was conducted both to identify potential barriers to participation and to understand the feasibility of various recruitment procedures. Benefits and limitations of ‘opt-out’ and ‘opt-in’ specimen collection methods were examined. Lastly, we developed study documents, including recruitment letters and informed consent forms, for future saliva and blood specimen collection efforts. Results: Two of the CRN HMOs currently have large-scale banked blood or saliva specimens (>20,000) within existing repositories. One site has two large-scale studies (>20,000 participants each) with banked specimens able to provide DNA for future research. Whole blood or blood components are the most commonly collected specimen types, and multiple CRN sites have access to banked tumor blocks. There were 33 past or current studies that included biospecimen collection. There was a trade-off between intensive data collection requirements and recruitment yield, cost, and participation burden. Conclusions: There is great potential for collection, storage, and use of biospecimen samples for research within the CRN. For future collection efforts, ‘opt-out’ models allow fast sample accrual, but impact data de-identification requirements. ‘Opt-in’ models allow for broad data collection, but are resource and time intensive. Pathology time and support may be a barrier to future use of stored samples.

Young pregnant women's views on the acceptability of screening for chlamydia as part of routine antenatal care

BackgroundIn pregnancy, untreated chlamydia infection has been associated with adverse outcomes for both mother and infant. Like most women, pregnant women infected with chlamydia do not report genital symptoms, and are therefore unlikely to be aware of their infection. The aim of this study was to determine the acceptability of screening pregnant women aged 16-25 years for chlamydia as part of routine antenatal care.MethodsAs part of a larger prospective, cross-sectional study of pregnant women aged 16-25 years attending antenatal services across Melbourne, Australia, 100 women were invited to participate in a face-to-face, semi structured interview on the acceptability of screening for chlamydia during pregnancy. Women infected with chlamydia were oversampled (n = 31).ResultsWomen had low levels of awareness of chlamydia before the test, retained relatively little knowledge after the test and commonly had misconceptions around chlamydia transmission, testing and sequelae. Women indicated a high level of acceptance and support for chlamydia screening, expressing their willingness to undertake whatever care was necessary to ensure the health of their baby. There was a strong preference for urine testing over other methods of specimen collection. Women questioned why testing was not already conducted alongside other antenatal STI screening tests, particularly in view of the risks chlamydia poses to the baby. Women who tested positive for chlamydia had mixed reactions, however, most felt relief and gratitude at having had chlamydia detected and reported high levels of partner support.ConclusionsChlamydia screening as part of routine antenatal care was considered highly acceptable among young pregnant women who recognized the benefits of screening and strongly supported its implementation as part of routine antenatal care. The acceptability of screening is important to the uptake of chlamydia screening in future antenatal screening strategies.

Application of a multiplex PCR to cervical cells collected by a paper smear for the simultaneous detection of all mucosal human papillomaviruses (HPVs) and typing of high-risk HPV types 16 and 18

A simple paper smear (PS) method for dry collection and storage of cervical specimens was employed to develop an easy multiplex (MPX) PCR for simultaneous detection of generic human papillomaviruses (HPVs) as well as typing of the high-risk HPV-16 and -18, the two clinically most important HPV genotypes, which are responsible for more than 80 % of cervical cancers. Multiplexing was performed with a small amount of DNA eluted by boiling from a single PS punch in a single tube and using a mixture of four pairs of primers specific for the HPV L1 consensus sequence, HPV-16, HPV-18 and the β-globin gene. Sixty HPV-positive biopsies and corresponding PS specimens from cervical cancer patients as well as cervical smears from 100 healthy women with or without abnormal cytology were collected both as PSs and in PBS. Detection of HPV DNA from cervical biopsies collected in PBS and corresponding cervical scrapes on a PS or in PBS by conventional and MPX-PCR showed a concordance of 100 % and adequacy of 93 %. A similar comparative study in cervical scrapes from normal women also revealed 100 % concordance. The technique was validated in a multicentric study at four different national laboratories. PSs collected by different centres showed variable adequacy (73-82 %) but the use of multiple PS discs for DNA extraction significantly increased the adequacy. Integration of PSs with MPX-PCR for the detection and typing of HPVs is a highly convenient, efficient, simple and cost-effective method for large-scale clinico-epidemiological studies and is also suitable for HPV vaccine monitoring programmes in resource-poor settings.

Fluorescence in situ hybridization for detecting urothelial carcinoma

Because urothelial carcinoma (UC) is associated with a significantly high risk of disease recurrence and progression, patients with UC require long-term surveillance. Fluorescence in situ hybridization (FISH) has been shown to be more sensitive than cytology in the detection of UC. The current study evaluated the use of FISH for detecting UC. A pathology database was used to identify patients who had urine cytology and FISH performed at the study institution between 2004 and 2006. Urinary specimens were analyzed using UroVysion FISH probes for abnormalities in centromeric chromosomes 3, 7, and 17 and locus-specific 9p21. FISH results were correlated with cytologic findings and a minimal clinical follow-up of 24 months. A total of 1006 consecutive urinary specimens from 600 patients (448 men and 152 women) who were monitored for recurrent UC (915 specimens) or evaluated for urinary symptoms (91 specimens) were identified. On FISH analysis, 669 specimens were found to be negative for UC and 272 specimens were positive for UC. Sixty-five (6%) specimens were insufficient for FISH analysis. The sensitivity and specificity of FISH for UC were 58% and 66%, respectively, and 59% and 63%, respectively, when FISH and cytology results were combined. Factors contributing to decreased FISH sensitivity included the paucity or absence of tumor cells, low-grade tumors, degenerated cells, method of specimen collection, type of specimen, and obscuring inflammatory cells or lubricant. UroVysion FISH appeared to have good sensitivity and specificity for detecting UC in urinary specimens. It is important to correlate the FISH results with the cytologic findings.

Non-invasive and simple methods for sampling rabbit DNA for PCR analysis of melanocortin 1 receptor (MC1R) gene mutations: a technical note

Simple and non-invasive methods of collecting and preserving biological specimens for genetic analysis can be useful in reducing the discomfort of the involved animals and can be applicable for highthroughput studies that require a large number of biosamples. In order to have the possibility to analyse a large number of samples and making easier the collection of rabbit biological materials as well as to reduce the cost and time of the genotyping procedures, here we tested different protocols for the genotyping of mutations identified in the rabbit melanocortin receptor 1 (MC1R) gene that are associated to coat colours in different rabbit breeds. These protocols make use of rabbit hair roots or cell preparations from buccal swabs with a direct PCR amplification without previous DNA extraction. Hair samples were collected from 60 rabbits of different breeds and preserved for up to 12 months at 4°C. For each hair sample 5-10 hair roots were clipped a few mm from the root directly within a 0.2 ml PCR tube. Buccal cells were collected from 10 crossbred and purebred rabbits and prepared after a few steps for PCR. The PCR fragments were analysed by means of polyacrylamide gel electrophoresis and stained with ethidium bromide or using fluorescent labelled products resolved in a capillary automatic sequencer. The success genotyping rate of the hair and buccal cell samples was 98% and 80%, respectively. These values are comparable to the success rates of other protocols described in human or other animals involving previous DNA extraction. The obtained results show that noninvasive methods of biological specimen collection that make it possible to analyse the DNA without previous extraction can be easily and routinely applied in rabbit molecular genetic studies. However, due to the higher success rate and the easier method of collection, plucked hairs seem the best source for high-throughput biosampling in this species.

The Role of Hysteroscopic Biopsy in Obtaining Specimens for Cytogenetic Evaluation in Missed Abortion prior to Suction Dilatation and Curettage

Aim:To estimate whether hysteroscopic-guided biopsy of gestational sac(s) in first trimester missed abortion increases the sensitivity of detecting aneuploidy compared to washing and careful specimen collection after suction dilatation and curettage (D&C). Materials and Methods:Thirty-five patients with first trimester missed abortion of which 25 underwent 29 suction D&Cs and 10 underwent hysteroscopic-guided biopsy of 12 gestational sacs prior to suction D&C. The karyotype of products of conception specimens were analyzed by G-banding techniques. Results:The percentage of specimens with 46,XX, 46,XY and aneuploidy were not significantly different in the hysteroscopic group [4/12 (33.3%), 3/12 (25.0%) and 5/12 (41.7%)] compared with the D&C group [8/29 (27.6%), 5/29 (17.2%) and 16/29 (55.2%)]. Although parity differed significantly between groups, it did not hold in a multivariable logistic regression model built to estimate whether the parity, gestational age and specimen collection method predict the likelihood of detecting a 46,XX chromosomal complement. Conclusions:Direct hysteroscopic-guided biopsies of gestational sac(s) do not increase the sensitivity of conventional cytogenetics for detecting aneuploidy when compared to specimens obtained by washing and microscopic identification of villi or fetal tissue after suction D&C.

Insights into semen analysis: a Chinese perspective on the fifth edition of the WHO laboratory manual for the examination and processing of human semen

Dear Editor, We are very glad to see that the Asian Journal of Andrology published a Special Issue on Semen Analysis in 21st Century Medicine, which well revealed some behind-the-scene controversies of the 5th edition of the WHO laboratory manual for the examination and processing of human semen 1. Three articles from the special issue, two on the reference values of semen parameters 2, 3 and another presenting the investigation results of 118 laboratories performing semen analysis in Mainland China 4, are very thought-provoking and we would like to share some of our views on these topics. First, we noticed that the lower limit of the reference values of semen parameters in the 5th edition of the manual is reduced. Can this reduction reflect a genuine decline in semen quality? We are doubtful on this point. The reasons for the reduction vary from the variations in population selection, and the biases in methodologies of measurement and data processing to the recommended application of 'stricter criteria'. It has been a long time since the change in semen quality of healthy men become a controversial issue throughout the world. There is no definite conclusion on the trend of overall semen quality, owing to the differences in study design, definition of normal men, specimen collection methods, methods used for measurement of sperm and for statistical analysis, selection biases, measurement errors, regional differences, and other factors such as age, years spent in semen collection, the period of abstinence, etc. In addition, researches on change of semen quality are only retrospective analyses. Various insurmountable biases and confounding factors also make the conclusion poorly reliable. Although careful efforts were made to reduce the biases in some studies, discrepancies in results still remained. With the publication of the 5th edition of the WHO manual, more concerns and debates would be aroused. So far, it is not yet known whether the reduced lower limit of the reference values can indicate the decline in semen quality, but strictly evidence-based study design, standardization of measurement criteria and quality control (QC) of semen analysis would explain the reduction in the lower limit. Second, we think it is necessary to establish the reference intervals for values of semen parameters based on a fertile population using the time-to-pregnancy method in China. Semen quality is commonly taken as a surrogate indicator of male fertility, reproductive toxicology, epidemiology and pregnancy risk assessments. Reference intervals for values of semen parameters from a fertile population and the standardization of procedures for the examination of human semen given in the 5th edition of the manual provide a realistic basis for medical professionals all over the world, especially in China, where more than one-fifth of the world population inhabits. China is in urgent need of reference values of semen parameters derived from fertile Chinese men, which calls for a prospective, multi-center and nation-wide population-based study. Such a large-scale study will provide evidence for the prognosis of fertility, diagnosis of infertility and impact of environmental factors on human existence. It will also be helpful for the State Food and Drug Administration of China to establish medical guidelines for male fertility regulation. Third, internal and external QC of semen analysis need to be further improved. Semen analysis is used widely and has a significant role in the assessment of male fertility. There have been reports on the absence of standardization on semen analysis 5, 6, 7, 8 and obvious differences among laboratories 9, 10, 11, and some researchers have called for a comprehensive QC 12, 13, 14, 15, 16. The lack of consistency and standardization 17 incurs difficulties for clinicians to interpret or compare the results of semen analysis from different laboratories. Therefore, improvements in the following aspects would help improve the quality of semen analysis and ensuring the reliability of the results: using accredited and standard measurement, implementing internal QC and external QC. Finally, the criterion for determining an eligible donor in China should be revised accordingly to elevate the present poor eligibility after comprehensive investigations. The current quality requirements for an eligible donor's semen as per the Essential Criterion and Technical Specification of the Human Sperm Bank, issued by the Ministry of Health of China, are based on the 4th edition manual. With the reduction in certain semen parameters such as semen concentration, motility and semen volume in the 5th edition, we believe that studies on the reference intervals for values of semen parameters from fertile men must be carried out before any modification of the criteria is made in China. Such expanded studies would lead experts to conduct extensive discussions and would thus provide informative suggestions for policy makers before making any modification in the criteria of the human sperm bank in China.

Swab and aspiration specimen collection methods and antibiogram in chronic suppurative otitis media at Jos University Teaching Hospital: Which is superior?

Chronic suppurative otitis media is a very common otologic problem in our environment. Appropriate methods for obtaining sample specimens for specific bacteria isolation has generated a lot of controversy. The simplest method available in our environment is the traditional swab method which, however, has been condemned on the basis of introducing contaminants. The objectives of this study were to compare the bacterial yield and the antibiogram of two specimen collection methods: the traditional swab method and aspiration method. This was a 3-month prospective study involving outpatients seen at both the emergency and outpatients' clinics of the Jos University Teaching Hospital in the period between May 2008 and July 2008. The biodata, duration of discharge and sites of samples were recorded in the study data form after obtaining consent from the patients or the parents of child. Eighty patients were studied comprising 40 each for aspiration and swab technique. This consists of 30 males (37.5%) and 50 females (62.5%) with a male to female ratio of 1:1.7. There were 24 (30%) children (14 males, 10 females). Six (4 males, 2 females) and 74 (26 males, 48 females) patients had bilateral and unilateral ear discharges, respectively. A total of 86 specimens were obtained in all, consisting of 42 left and 44 right ears. There were 68 bacteria isolates comprising Pseudomonas (30), Staphylococcus (18), Proteus (12), and (8) Klebsiella species. Two were incidental fungal isolates of (Candida species), (8) cultures grew contaminants and (10) specimens had no growth at all. Each of the swab and aspiration techniques had (44) specimens. Despite the controversy surrounding the sampling technique in literature, swab technique has been found to be as good as the aspiration technique in our study. The organisms isolated are the same as those obtained in other places. Contaminants found were few and occurred in equal amount in the same patients in the two methods.