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PubMedDate not supplied

The need for three stool specimens in routine laboratory examinations for intestinal parasites.

This study was designed to test whether three sequential stool specimens are necessary for reliable detection of intestinal parasites in routine laboratory examinations. There were 175 patients in whom a single species of intestinal parasite or ova was identified on at least one occasion when three stool specimens were examined over a period of 14 days from the first recorded specimen. Examination of a first stool specimen detected parasites and/or ova in 102/175 patients (58.3%); examination of a second specimen identified an additional 36 positive patients (20.6%) and examination of a third specimen yielded a final 37 previously undetected patients (21.1%). These findings indicate that at least three stool specimens should be examined to confirm the presence of intestinal parasites. Even with such practice, it is not possible to guarantee that there is no infestation, but examination of fewer specimens would run a definite risk of false negative results.

Feces↗
PubMed1993

Detection of Epstein-Barr virus DNA in sequential renal transplant biopsy specimens using the polymerase chain reaction. Report of a patient with a posttransplant lymphoproliferative disorder.

The polymerase chain reaction was used to retrospectively examine frozen and paraffin-embedded tissue from four sequential renal biopsy specimens, a pretransplant skin biopsy specimen, a postauricular lymph node, and an allograft nephrectomy specimen for the presence of Epstein-Barr virus (EBV) DNA in a 29-year-old man who developed diffuse lymphadenopathy 2 months after a cadaveric renal transplant. The EBV primers used amplify a 245-base pair fragment of the EBV IR3 sequence and were run in parallel with a pair of human beta-globin gene primers as positive DNA integrity controls. Epstein-Barr virus DNA was detected in three of the four renal allograft biopsy specimens, the post-auricular lymph node, and the renal allograft nephrectomy specimen. Initial detection occurred 34 days posttransplant and 23 days prior to the diagnosis of posttransplant lymphoproliferative disorder. Examination of a skin biopsy specimen obtained prior to transplantation and of the initial posttransplant renal allograft biopsy specimen 15 days posttransplant did not demonstrate EBV DNA. The findings of serologic studies performed at the time of diagnosis of the posttransplant lymphoproliferative disorder suggested a remote EBV infection. These results further demonstrate the association of EBV with posttransplant lymphoproliferative disorder and suggest a possible role for polymerase chain reaction in the early detection of EBV in transplant patients. Larger retrospective studies are needed to determine the prognostic significance of EBV DNA found in routine renal allograft biopsy specimens.

Adult↗
PubMed1996

Modified clearing technique to identify lymph node metastases in post-irradiated surgical specimens from rectal adenocarcinoma.

The aim of this study is to describe a modification of the original method of clearing lymph nodes in specimens from rectal adenocarcinoma and its yield in comparison to a control group whose specimens were studied by a manual technique. There were 32 consecutive patients with locally advanced rectal adenocarcinoma. All patients received preoperative radiation therapy (PRT) at doses of 45 Gy, concomitant with 5-fluorouracil at the doses of 450 mg/m2 IV bolus, days 1-5 and 28-33 of PRT; 4-8 weeks later radical surgery with a curative intent was performed. Twenty two specimens were studied by a manual technique consisting of fixation in 10% formalin. Ten specimens were studied with the modified clearing technique consisting of administration through the inferior mesenteric artery of methylene blue; fixation with 10% formalin plus 0.01% of methylene blue; clearing the fat with a sequence of 95% alcohol-100% acetone-100% xylene. In the 32 specimens, 413 lymph nodes were found. Using the manual technique 104 lymph nodes were found (average 4.7 lymph nodes per specimen); 309 lymph nodes by the modified clearing technique (average 30.9 lymph nodes per specimen), (p < 0.0001). Using the manual technique 23 out of 104 lymph nodes (22%) contained metastases. All metastatic lymph nodes measured > 5 mm; 18 out of 23 metastatic lymph nodes were located on the perirectal area (level 2); and five were located in the root of the inferior mesenteric artery (level 5). Using the modified clearing technique 278 out of 309 (89%) lymph nodes measured < 5 mm; 15 out of 309 (4.8%) lymph nodes contained metastases; 9 out of 15 (60%) measured > 5 mm and were located on levels 2 and 5; and six, located on level 5, measured < 5 mm. The modified clearing technique is a quick, easy and reproducible method for identifying lymph nodes in post-irradiated surgical specimens. In comparison to the traditional manual method of searching for lymph nodes, it improves in the detection of small lymph nodes (< 5 mm), allowing better staging in patients with rectal adenocarcinoma.

Adenocarcinoma↗
PubMed1997

Identification of unique cocaine metabolites and smoking by-products in postmortem blood and urine specimens.

Toxicological investigation of suspected cocaine-related deaths routinely involves the identification of cocaine (COC) and its metabolites including benzoylecgonine (BE) and ecgonine methyl ester (EME) in postmortem specimens. We utilized solid-phase extraction followed by gas chromatography/mass spectrometry for the qualitative and quantitative analysis of cocaine and eight cocaine-related analytes. These analytes included anhydroecgonine methyl ester (AEME), a unique product formed during cocaine smoking, and cocaethylene (CE), formed by transesterification of cocaine in the presence of ethanol. Thirteen pairs of postmortem heart blood and urine specimens were analyzed from cases of death due to acute cocaine intoxication, multiple drug intoxication, or other non-drug related causes. COC, EME, and BE were detected in all specimens. The range of concentrations in blood were: COC, 23-2088 ng/mL; BE, 215-9195 ng/mL; and EME, 220-7275 ng/ mL. AEME was identified in 2 blood and 10 urine specimens, and CE was identified in 1 blood specimen and 4 urine specimens. The identification of AEME in the specimens indicated that "crack" cocaine had been smoked, and the presence of CE indicated co-administration of cocaine and ethanol. The presence of these unique cocaine analysis in postmortem specimens provides valuable information regarding the cause and manner of death.

Cocaine↗
PubMed1998

Cerebrospinal fluid cytology: an 11-year experience with 5951 specimens.

OBJECTIVE: Cerebrospinal fluid (CSF) diagnoses encompass a wide spectrum of conditions. The authors review one institution's CSF cytology results over an 11-year period. DESIGN AND SETTING: A retrospective study of 5951 CSF specimens generated between 1985 and 1995. Specimens from pediatric patients (<19 years of age) from the same time period were separately identified. RESULTS: A total of 5561 adult and 390 pediatric CSF specimens were interpreted. A diagnosis of "negative for malignant cells" was assigned in 5171 (93%) of the adult cases and in 351 (90%) of the pediatric cases. Specific infectious organisms were identified in 26 adult specimens and one pediatric specimen. Cryptococcus was the most common infectious agent observed (n = 23 adults), and Toxoplasma was the sole pediatric infectious agent. Two hundred seventy-six (5%) adult cases and 31 (8%) pediatric cases were positive for malignant cells. Diagnoses included metastatic tumors (adult, 140 [51%]; pediatric, 0); lymphoma/leukemia (adult, 112 [41%]; pediatric, 4 [13%]); malignant unclassified neoplasms (adult, 9 [3%]; pediatric, 0); and primary central nervous system neoplasms (adult, 12 [4%]; pediatric, 27 [87%]). Medulloblastoma was the most common pediatric neoplasm (n = 21). There were 105 (2%) adult cases and 8 (2%) pediatric cases with atypical cells present. Atypical lymphoid cells were the most common type in adult cases (53%). CONCLUSIONS: In our experience, infectious agents were rarely identified in pediatric CSF specimens. In adult specimens, the most commonly identified organisms was Cryptococcus. Primary central nervous system neoplasms accounted for a higher percentage of CSF specimens in the pediatric population than in the adult population. The most commonly identified malignancy in adults was metastatic neoplasms, and in children, medulloblastoma.

Adenocarcinoma↗
PubMedDate not supplied

The expression of C-myb in human metastatic melanoma cell lines and specimens.

The rapidly increasing incidence of malignant melanoma and lack of effective systemic therapy for advanced disease has given rise to the need for new approaches. The suggestion that c-myb may be an important gene in the control of melanoma proliferation prompted the exploration of its expression in several metastatic melanoma cell lines and specimens. Initial Northem hybridization showed undetectable expression of c-myb in the cell lines, although it was very strongly expressed in the K-562 cell line. Therefore, c-myb expression was examined utilizing primers and RT-PCR on the five melanoma cell lines and thirty-two metastatic melanoma specimens. There was very low expression in the two cell lines (UISO Mel-1 and 3) that were nontumorigenic, whereas there was a 10 fold increase in expression in the tumorigenic cell lines. In the metastatic specimens the expression varied by over 100 fold between the lowest and highest specimens. The expression of c-myb in tumor specimens was in general greater than matched normal specimens, save for skin which had moderate expression. In general, there did not appear to be any correlation between the clinical characteristics of the various specimens and the amount of c-myb expression. However, females with lymph node metastases had somewhat lower expression than males. The fact that significant melanoma specimens have altered expression of c-myb, coupled with the previous inhibition of melanoma growth by antisense c-myb, suggests that there may be a potential role for c-myb antisense therapy in the treatment of this tumor.

Female↗
PubMed1997

Biological and environmental specimen banking at the Centers for Disease Control and Prevention.

Scientific programs at the Centers for Disease Control and Prevention encompass diverse public health interests. These programs include investigations of newly emerging infectious diseases, assessments of chronic disease risk factors, and evaluations of environmental health hazards. Since the early 1960s, CDC has maintained a specimen bank to retain aliquots of biological specimens collected from a variety of epidemiologic investigations as well as from the National Health and Nutrition Examination Surveys (NHANES). CDC's National Institute of Occupational Safety and Health (NIOSH) also maintains a repository of environmental materials from its investigations. To extend its repository capabilities more effectively, CDC has begun developing a new facility that, when finished, will meet CDC's storage needs for both biological and environmental specimens. A highly complex but very flexible information management system for this project, enabling the storage of data related to studies for which these specimens were originally collected, has already been completed. Proper specimen collection, archiving, and short- or long-term storage of specimens for environmental health-related analyses is critical for the work of the staff of the Division of Environmental Health Laboratory Sciences, National Center for Environmental Health, who perform a variety of biochemical analyses on biological specimens, including quantitation of dioxins, furans, coplanar PCBs, pesticides, volatile organic compounds, metabolites, essential and toxic trace elements, vitamins, and lipids, and also conduct genetic screening. Information regarding these analytes is an essential part of the CDC Repository Database.

Biological Specimen Banks↗
PubMed2003

Stability of archived liquid-based cervical cytologic specimens.

BACKGROUND: Exfoliated cervical cell specimens collected in PreservCyt, a methanol-based medium used in ThinPrep liquid-based cytology, have been archived in epidemiologic studies. However, long-term DNA stability and cytologic stability of these biospecimens have not been evaluated. METHODS: Cervical specimens were collected into PreservCyt from participants in a natural history study of human papillomavirus (HPV) infection and cervical carcinoma in Guanacaste, Costa Rica (1993-2000), and stored at ambient temperatures. Thirty specimens classified as low-grade squamous intraepithelial lesions by liquid-based cytology were randomly chosen from each collection year (except for 1994) and selectively assessed for molecular and cytologic stability. Specimens were tested in 2001 for 1) HPV DNA by the Hybrid Capture 2 test, 2) beta-globin DNA by polymerase chain reaction amplification of multiple length fragments (268, 610, and 1327 bp), and 3) nuclear preservation by visual inspection of newly made liquid-based cytology slides. All testing was done masked to year of collection. Associations of stability and storage time were evaluated using standard contingency tables and chi-square tests for trend. RESULTS: Human papillomavirus DNA, as detected by the Hybrid Capture 2 test, was unaffected by storage time. Stability of beta-globin DNA (P(Trend) < 0.0001) and nuclear preservation (P(Trend) < 0.0001) declined with increasing storage time. Approximately 15% of specimens could not be amplified for any beta-globin DNA fragment after 5 years of storage (collected in 1996). In addition, cytology slides made from 41% specimens were rated as marginal (32%) or unsatisfactory (9%) after 8 years of storage (collected in 1993). CONCLUSIONS: Cervical specimens archived in PreservCyt underwent partial DNA and cytologic degradation after several years of storage. Methodologic studies to optimize long-term storage of cervical cells for epidemiologic studies of cervical carcinoma are needed.

Adult↗
PubMed2003

Application of laser capture microdissection to cytologic specimens for the detection of immunoglobulin heavy chain gene rearrangement in patients with malignant lymphoma.

BACKGROUND: The demonstration of the monoclonality of immunoglobulin heavy chain (IgH) gene rearrangement is an indispensable method for the diagnosis of B-cell lymphoma as well as histocytochemical analysis. For the detection of IgH gene rearrangement, the extraction of DNA from a homogenous cell population is necessary. Recently, the laser capture microdissection (LCM) technique was shown to isolate specific cells from histopathologic specimens for molecular analysis. However, to the authors' knowledge the applicability of LCM to cytologic specimens has not yet been well established. METHODS: Using LCM, a homogenous population of B-cell lymphoma cells as both histologic sections and cytologic specimens was captured, and genomic DNA was extracted from the captured cells. IgH gene rearrangement was analyzed by the polymerase chain reaction (PCR)-based single-strand conformational polymorphism (SSCP) method. RESULTS: Genomic DNAs were extracted successfully from ethanol-fixed cytologic specimens, but cells were not captured from air-dried specimens. Using PCR-SSCP analysis, the monoclonality of the IgH gene rearrangement was detected in five cases of tissue sections among nine analyzed cases of malignant lymphoma diagnosed immunohistochemically. However, analysis of the cytologic specimens with LCM demonstrated the monoclonality of the IgH gene rearrangement in seven cases of lymphoma. CONCLUSIONS: The results of the current study suggest that the novel application of LCM to cytologic specimens occasionally exhibits high sensitivity for the detection of IgH gene rearrangement monoclonality compared with the use of histologic sections.

Aged↗
PubMed1991

DNA slit-scan flow cytometry of bladder irrigation specimens and the importance of recognizing urothelial cells.

DNA slit-scan flow cytometry was used to analyze 150 bladder irrigation specimens from 83 patients. Specimens were categorized into groups based on cystoscopy, histology, and cytopathology. Cells were stained for DNA with propidium iodide using a whole cell protocol. Non-specific fluorescence in the cytoplasm of some urothelial cells together with differential DNA staining of cell types in certain specimens was noted. DNA frequency distributions were analyzed using a semi-automated technique. Data were gated using slit-scan morphological features to remove cellular debris, multiple nuclei, and cells exhibiting nonspecific cytoplasmic fluorescence. Specimens were classified abnormal if they were aneuploid or had a hyperdiploid fraction (HDF) greater than 8%. The sensitivity to abnormality was 89% for grade 3 transitional cell carcinoma (TCC), 70% for grade 2 TCC, and 67% for grade 1 TCC. Specificity was 61%. Specimen data were then reprocessed using slit-scan morphological features to enrich for urothelial cells. The urothelial cells were identified by the ratio of nuclear diameter to cell diameter. This method was found to be in good agreement with immunofluorescent labeling of urothelial cells using the urothelium-selective T16 monoclonal antibody. The sensitivity to abnormality remained 89% for grade 3 TCC and 70% for grade 2 TCC, but fell to 52% for grade 1 TCC. Specificity for the urothelial cell enriched data increased to 77%. Reprocessing of data to enrich for urothelial elements resulted in 16 fewer specimens with an aneuploid DNA distribution and 2 fewer specimens with increased HDF.(ABSTRACT TRUNCATED AT 250 WORDS)

Aneuploidy↗
PubMed1985

Biomechanical time-tolerance of fresh cadaveric human spine specimens.

Changes in the biomechanical properties of fresh cadaveric spinal specimens due to long-term freeze storage and long test periods have been investigated. Fresh cadaveric specimens were divided into three groups: Group A specimens were tested fresh on the 1st day and 13 subsequent days; Group B specimens were tested on the 1st day, frozen in sealed bags at -18 degrees C for 21 days, and tested for 13 consecutive days after thawing; and Group C specimens were frozen for up to 232 days and tested for 14 consecutive days after thawing. We could not find any significant differences between the behavior of the three test groups. This implies that freezing and storage, even for long periods, do not significantly alter the physical properties of cadaveric spinal specimens. Concerning the differences observed on a daily basis, the mean value of the maximum displacement for the 1st day did not differ significantly from the corresponding mean value for the 13 consecutive days. This was true for all three groups, although there was some indication that the fresh group specimens showed greater variation than the two frozen groups.

Adult↗
PubMed1978

Bacteriuria in the puerperium: an evaluation of methods for collecting urine specimens.

Five hundred and eighteen puerperal patients were screened for bacteriuria by examination of clean-catch, midstream urine specimens using the dip-slide cultivation technique. Bladder urine from 105 patients showing 10(4) or more bacteria per milliliter of midstrean urine was obtained by suprapubic bladder aspiration and cultivated, along with two consecutive midstream specimens. The bacteriologic results in these specimen categories are compared. Of 23 patients showing growth in the midstream samples compatible with a true bacteriuria, 18 had sterile puncture urine specimens. Consecutive contaminated midstream specimens from the same patient usually yielded growth of the same bacterial species. It is concluded that a diagnosis of bacteriuria in the puerperium cannot be based upon cultural results of midstream specimens alone. In several of the bladder urine specimens, microogranisms being nonpathogenic in the urinary tract and residing normally in the vaginal flora were found, probably due to the traumatic effect of partus. In Oslo the frequency of bacteriuria among women post partum is estimated at a maximum of 1 per cent.

Bacteriuria↗
PubMed2005

Evaluation of separate vs pooled swab cultures, different media, broth enrichment and anatomical sites of screening for the detection of methicillin-resistant Staphylococcus aureus from clinical specimens.

Early identification of methicillin-resistant Staphylococcus aureus (MRSA) carriers is a major component of an MRSA control programme. The cost and laboratory workload could be markedly reduced by processing multiple swabs from one person in one culture broth (specimen pooling). We evaluated the sensitivity for MRSA detection and the growth rate of pooled swabs compared with individual processing. In total, 1254 swabs from 423 subjects (two to five swabs per subject) were submitted for detection of MRSA. Swabs were suspended in 2-mL volumes of sterile Todd-Hewitt Broth and divided into two 1-mL aliquots. One aliquot of the suspension was processed as a single specimen, and the other aliquot was mixed (pooled) with other suspensions in which swabs from the same patient were suspended. Forty-four (10%) pooled samples were positive for MRSA. Specimens from seven additional patients that were negative when pooled were positive when processed separately. There was no case where the pooled specimen was positive but the separate specimens were negative. The diagnostic sensitivity of pooled surveillance cultures compared with single cultures, when only subjects colonized by MRSA were considered, was 86% and the false-negative rate was 14%. Eighty percent of the pooled positive cultures were detected by the third day and all were detected by the fourth day. Fifty-four percent of the specimens processed separately were detected by the second day and all were detected by the fourth day. Pooling of specimens decreases the sensitivity of MRSA detection compared with processing each swab separately, particularly in swabs with a low number of colony-forming units. In all subjects whose pooled samples were negative but whose swabs examined separately were positive, the swabs examined separately were negative on primary plates and positive only after culturing in enrichment broth.

Axilla↗
PubMed2006

Examination of laparoscopic retrieval bag washings for malignant cells after hand-assisted laparoscopic radical nephrectomy and intact specimen removal.

OBJECTIVES: Port site metastases after hand-assisted laparoscopic radical nephrectomy have been described in the literature. There is no uniform agreement among urologists regarding the use of a retrieval bag before intact specimen removal. The aim of this study was to determine whether LapSac renal extraction bag washings contain malignant cells. METHODS: We prospectively obtained washings from the LapSac retrieval bag after hand-assisted laparoscopic radical nephrectomy and intact specimen removal for renal cell carcinoma. In 30 consecutive cases, after removal of the kidney specimen from the LapSac, the LapSac was irrigated with 50 mL sterile Hank's balanced salt solution. These washings were sent for cytologic examination. Cytologic evaluation was performed with a Thin Prep and the Papanicolaou method. RESULTS: We performed 30 hand-assisted laparoscopic radical nephrectomies for suspected renal cell carcinoma with the above protocol. One specimen was benign and one showed transitional cell carcinoma; these were excluded from the study. Six specimens were stage T1a, 17 were T1b, 1 was T2, 2 were T3a, and 2 were T3b. Histopathology revealed 27 specimens with clear cell renal cell carcinomas with Fuhrman grades from 1 to 4; 1 specimen showed chromophobe renal cell carcinoma. Margins were negative in all cases, and there were no gross or microscopic tumor violations. The cytologic results from 27 cases were negative and in 1 case with T3b renal cell carcinoma the LapSac washings were positive for malignant cells. CONCLUSIONS: The preliminary findings from our study show that low-stage, low-grade tumors removed laparoscopically with minimal manipulation do not exfoliate cells into their LapSac retrieval bags.

Carcinoma, Renal Cell↗
PubMed1988

Diagnosis of nosocomial bacterial pneumonia in intubated patients undergoing ventilation: comparison of the usefulness of bronchoalveolar lavage and the protected specimen brush.

PURPOSE: To compare the usefulness of specimens recovered using a protected specimen brush and those recovered by bronchoalveolar lavage in the diagnosis of nosocomial pneumonia occurring in intubated patients undergoing ventilation, we performed both procedures in patients suspected of having pneumonia because of the presence of a new pulmonary infiltrate and purulent tracheal secretions. PATIENTS AND METHODS: Twenty-one patients (16 men and five women) with an average age of 57 +/- 12 years were studied. They had been receiving mechanical ventilation for 8 +/- 6 days before inclusion in the trial. The clinical suspicion for nosocomial bacterial pneumonia was high in these patients. Fiberoptic bronchoscopy was performed in each patient. Bronchoscopy specimens were obtained by a protected specimen brush and by bronchoalveolar lavage, and were then processed for quantitative bacterial and fungal culture using standard methods. Total cell counts were performed on an aliquot of resuspended original lavage fluid. Differential cell counts were made on at least 500 cells. In addition, 300 cells were examined at high-power magnification and the percentage of cells containing intracellular microorganisms and the average number of extracellular organisms per oil-immersion field were determined. RESULTS: Quantitative culture of specimens recovered using the protected specimen brush were positive (more than 10(3) colony-forming units [cfu]/ml) in five of five patients with subsequently confirmed pneumonia, and negative (less than 10(3) cfu/ml) in 13 of 13 patients without bacterial pneumonia, but results were not available until 24 to 48 hours after the procedure. Quantification of intracellular organisms in cells recovered by lavage was also useful in distinguishing patients with pneumonia (more than 25 percent of cells with intracellular organisms in five of five patients) from those without pneumonia (less than 15 percent of cells with intracellular organisms in all cases), and results were available immediately. In contrast, quantitative culture of lavage fluid and differential cell counts were of little value in identifying infected patients. CONCLUSION: The protected specimen brush and microscopic identification of intracellular organisms in cells recovered by lavage yield useful and complementary information, and together permit rapid and specific treatment of most patients with nosocomial pneumonia.

Bacteria↗
PubMed1999

Routine use of the Gen-Probe MTD2 amplification test for detection of Mycobacterium tuberculosis in clinical specimens in a large public health mycobacteriology laboratory.

The new version of the Amplified Mycobacterium Tuberculosis Direct Test, MTD2 (Gen-Probe Inc., San Diego, CA) has been implemented as part of the regular testing algorithm for detecting Mycobacterium tuberculosis (MTB) in selected respiratory and non-respiratory specimens in our laboratory. At the Central Public Health Laboratory, Etobicoke, Ontario, we receive specimens for the detection of mycobacteria from all areas of the Province of Ontario. The laboratory processes approximately 25,000 specimens per year, and receives approximately 2000 reference cultures for identification. There are 600 to 700 new cases of tuberculosis detected yearly. Over the 1-year period (1997-98), 823 specimens were tested by MTD2 and the results were compared with radiometric culture (Bactec, Becton Dickinson, Sparks, MD) and clinical diagnosis, giving an overall sensitivity, specificity, positive predictive value and negative predictive values of 100%, 99.6%, 97.4% and 100%, respectively. Two hundred and two cases of respiratory TB and 56 cases of extrapulmonary TB were detected by MTD2 within 0-4 days of specimen arrival in the laboratory. By appropriate selection of specimens for testing, the MTD2 can provide a fast, accurate, and cost-effective method for the detection of MTB in clinical specimens.

Bacteriological Techniques↗
PubMed2004

Optimization of PCR based detection of human papillomavirus DNA from urine specimens.

BACKGROUND: Human papillomavirus (HPV) causes cervical cancer. Current screening requires a yearly pelvic exam and Pap smear. However, these procedures are impractical for screening all women at risk for disease. Urine sampling has been successfully utilized to screen for Chlamydia trachomatis (CT) and Neisseria gonorrhoreae (NG) infections and has been considered for HPV DNA detection by several investigators. However, no study to date has been performed to specifically optimize HPV detection in urine. OBJECTIVES: To compare handling and extraction techniques in order to optimize the HPV specific PCR system in urine specimens. STUDY DESIGN: Examination of 10 characteristics that may contribute to PCR inhibition in urine was performed utilizing 10SG mulitstixs. Five different DNA extraction methods were compared in spiked specimens and in 10 clinical specimens. After the optimal extraction technique was identified, concentration of the sample with and without prior dilution was compared to the original protocol. Lastly, specimen handling was compared between immediate processing, refrigerating overnight, or freezing overnight. RESULTS AND CONCLUSIONS: the presence of protein in urine enhanced amplification while nitrites decreased amplification. Of the extraction methods tested, the QIAamp DNA Mini Kit demonstrated the best amplification from urine samples spiked with HPV DNA and clinical specimens. The addition of a dilution step and a concentration step before applying the Qiagen protocol further increased amplification of beta-globin (from 50 to 63%) and the HPV L1 gene (from 13 to 33%). Lastly, refrigerating the specimens at 4 degrees C overnight appears to produce better amplification (62% beta-globin and 17% HPV positive) than either immediate processing (46% beta-globin and 13% HPV+) or freezing the specimen for 24h prior to processing (46% beta-globin and 10% HPV+). In these studies, amplification was low despite optimization. Additional improvements are required prior to clinical application of a urine-based HPV DNA detection system.

DNA, Viral↗
PubMed2005

Importance of specimen size in accurate needle liver biopsy evaluation of patients with chronic hepatitis C.

BACKGROUND & AIMS: In patients with chronic hepatitis C (CHC), percutaneous needle liver biopsy examination establishes the severity of necroinflammatory activity and fibrosis, thus guiding treatment decisions. Optimal biopsy specimen size remains controversial. We sought to determine how varying lengths of biopsy specimens influence the grading and staging of CHC. METHODS: We used 100 liver biopsy specimens from patients with CHC. The slides were evaluated blindly using the METAVIR scoring system, after being covered with paper, so that only specific specimen lengths (5 mm, 10 mm, 15 mm, and > or =20 mm) were visible. In each case, the scores obtained with biopsies 5 mm, 10 mm, or 15 mm long were compared with the scores at 20 mm or greater by weighted kappa statistics (kappa of >.75 signified excellent agreement). A subset of specimens 20 mm or greater was selected for a blinded repeat scoring to assess intraobserver agreement. The kappa statistics for the designated features and lengths were compared using analysis of variance. RESULTS: In assessing the stage of fibrosis, the weighted kappa statistics for agreement with the 20-mm or greater score at 5 mm, 10 mm, and 15 mm were .75, .85, and .92, respectively. In assessing the histologic activity score, the corresponding figures were .73, .81, and .77, respectively. Average kappa statistic comparisons showed that intraobserver agreement was significantly better than agreement between the 20-mm or greater scores and those at shorter lengths; the 5-mm kappa scores were significantly lower than the others; and there was no significant difference between the 10-mm and 15-mm kappa scores. CONCLUSIONS: Liver biopsy specimens measuring at least 10 mm usually reflect the grade and stage of CHC reliably. Relatively little improvement in diagnostic accuracy is obtained with longer specimens.

Biopsy, Needle↗