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Microdissection and gene rearrangement analysis of paraffin-embedded specimens of orbital malignant lymphoma.

PURPOSE: To determine whether a definite diagnosis of malignant lymphoma can be made from paraffin-embedded archived orbital specimens by gene rearrangement analysis using microdissection and polymerase chain reaction (PCR). METHODS: Specimens from four patients with histopathologically diagnosed orbital malignant lymphoma were examined. The malignant cells were microdissected off the paraffin-embedded specimens. DNA was extracted from the cells, and the immunoglobulin heavy chain ( IgH) gene was amplified by PCR. Gene rearrangements were detected by using primers for the third framework (FR3A), the second framework (FR2A), and the complementary determining region 3 (CDR3). Translocation of the B-cell lymphoma/leukemia-2 ( bcl-2) gene was also examined. RESULTS: Malignant cells were present on the slides of the paraffin-embedded specimens of three of four cases. The specimens from these three cases showed IgH rearrangements for FR3A, FR2A, and CDR3. A bcl-2-associated translocation was also detected in one case. CONCLUSIONS: Gene rearrangement analysis is applicable to paraffin-embedded archived orbital specimens to confirm a diagnosis of malignant lymphoma. The advantage of this method is that only a small specimen is needed because the detection sensitivity is high.

Complementarity Determining Regions↗

Prevalence of papillomarvirus infection in colposcopically directed cervical biopsy specimens in 1972 and 1982.

A total of 1264 consecutive cervical biopsy specimens obtained at the Parkland Memorial Hospital Dysplasia Clinic during 1972 were reviewed. Histopathologic specimens were assessed with special reference to changes induced by human papillomavirus. In 1972, only 0.7% of biopsy specimens were reported as consistent with human papillomavirus infection. Upon review, however, 36.5% of these specimens were found to demonstrate histologic criteria for the diagnosis of human papillomavirus infection. Approximately half of biopsy specimens reclassified as human papillomavirus were originally interpreted as inflammation; the others were interpreted as cervical intraepithelial neoplasia. Patients with human papillomavirus infection were significantly younger than patients with cervical intraepithelial neoplasia (24.9 versus 30.2 years). These findings were compared with 965 cervical biopsy specimens obtained in 1982. Thirty-four percent of these biopsy specimens revealed human papillomavirus infection. These observations support the concept that human papillomavirus infection of the cervix is not a new entity but a previously unrecognized finding whose prevalence has been relatively stable over a 10-year period.

Adult↗

The underestimation of Young's modulus in compressive testing of cancellous bone specimens.

In order to determine the accuracy of measurements of Young's modulus of cancellous bone by conventional compression testing, two independent strain measurements were made simultaneously during non-destructive uniaxial compression to 0.8% strain of rectangular specimens (n = 18). Strain was measured by an extensometer attached to the compression anvils close to the specimen and by an optical system covering the central half of the specimens. Mean Young's modulus determined by the extensometer technique was 689 MPa, but was 871 MPa when determined by the optical technique (mean difference = 182 MPa, SED = 50 MPa, p less than 0.002). Uneven strain distribution due to lack of support of cut vertical trabeculae at the anvil-specimen interface is believed to be causing the underestimation of Young's modulus measured by the extensometer technique. The influence of friction at the specimen-anvil interface was studied by performing a finite element analysis. It is concluded that Young's modulus of specimens of the chosen geometry on average is underestimated by about 20% by conventional compressing testing. The underestimation seems not to be dependent upon specimen density.

Biomedical Engineering↗

Specimen findings and survival after preoperative 252Cf neutron brachytherapy for stage II cervical carcinoma.

Twenty-seven patients with operable Stage II cervical cancer with a mean diameter tumor of 8.0 cm were studied in a feasibility study using preoperative 252Cf implants plus whole-pelvis radiation to 45 Gy followed by extrafascial total abdominal hysterectomy and bilateral salpingo-oophorectomy 4 to 6 weeks later. Hysterectomy specimens were studied by a set protocol. With the protocol used, 13/27 (51%) specimens and abdominal stagings were negative for residual tumor. The survival rate for the patients with negative findings was 93% at 5 years. In 14/27 (49%) patients the specimens were positive for residual tumor. In contrast, the 5-year survival rate for this group was 46% (P less than 0.001). In these patients several interrelated factors were determined to be of importance, i.e., (1) tumor size greater than 8 cm in maximum diameter, (2) positive or negative residual tumor status, and (3) total dose of radiation given. Survival was lower for larger tumors and specimens were more likely to show residual tumor. A lower treatment dose led to more positive specimens, as well as to poorer survival. While the patients with Stage II disease fared very well when negative specimens were found, further prospective studies of the appropriate treatment for those with positive tumor specimens are needed.

Brachytherapy↗

Reactivity of five anti-HIV-1 subtype O specimens with six different anti-HIV screening ELISAs and three immunoblots.

Five anti-subtype O specimens were tested by anti-HIV-1/2 screening and confirmatory assays. They can be divided into three specimens, reactive with all ELISAs, independent of the nature of the antigen (recombinant proteins or peptides) and test configuration (indirect ELISA or double antigen/sandwich ELISA). One specimen was not detected by one peptide based ELISA. One specimen was only recognized by two ELISAs and should be considered as a marker sample for the weakness of currently used ELISAs with anti-subtype O. Three different immunoblot assays available commercially detected two of the specimens with a major binding of gp160 and other viral bands, especially the integrase and reverse transcriptase. Another two specimens lacked reactivity with glycoproteins almost completely, but showed some staining with the enzymes of HIV, and would most probably be interpreted as indeterminate. The fifth specimen, which was also missed by most of the ELISAs, had very faint staining of the gp160 and a very weak staining of p24, and would most probably be interpreted as negative. Adaption of currently available tests to anti-subtype O is needed for the future reliability of anti-HIV diagnostic reagents.

AIDS Serodiagnosis↗

Correlation of surface topography of metal-shadowed specimens with their negatively stained reconstructions.

We present a comparison of surface reconstructions from three different freeze-dried and unidirectionally metal-shadowed specimens (i.e. bacteriophage T4 polyheads, crystalline actin sheets and nuclear pore complexes) with two- or three-dimensional reconstructions of the same specimens when prepared by negative staining. Based on these and many published results, the following conclusions have been reached: With a "cooperative" specimen (e.g. the polyheads), the surface reconstruction computed from a metal-shadowed replica compares favorably with two- or three-dimensional reconstructions obtained from the same specimen after negative staining at the 3-4 nm resolution level. This relatively "poor" level at which the surface topographies of the two preparations can be compared appears to be set by a "practical" resolution limit (i.e. of distinct and reproducible structural detail) of metal replicas of biological specimens, despite the appearance of weak higher-order diffraction spots (i.e. corresponding to 2-3 nm). While in some cases (e.g. the crystalline actin sheets) the surface reliefs of metal replicas may bear little resemblance to the actual structure under investigation, the replicas may still contain sufficient features to establish the polarity or handedness of the structure (i.e., the "top" and "bottom" surfaces of a crystalline sheet). Information from negatively stained specimens is usually complementary with information from freeze-dried and metal-shadowed specimens. However, there are artifacts in both techniques, and we present an example with the nuclear pore complex, where these techniques yield confusing results.

Actins↗

Strategy for efficient detection of respiratory viruses in pediatric clinical specimens.

Direct immunofluorescence (IF) with a polyclonal respiratory syncytial virus (RSV)-specific antibody preparation was used for antigen detection during the 1982-1983 RSV season (155 specimens) and gave an overall sensitivity of 94% with 87% specificity compared with viral culture. Indirect IF was used in the 1983-1984 season (265 specimens) and exhibited sensitivity of 96% with specificity of 79%. During these two seasons, 42 of 224 (18.8%) specimens that were IF-negative for RSV grew viruses other than RSV. In the winter of 1984-1985, we screened 297 specimens for RSV by IF and 80 (27%) were positive. Forty-four (20%) of the IF-negative specimens were culture-positive for RSV(2) or other viruses(44). We conclude that, in the interest of cost reduction and expeditious detection of respiratory viruses, once a properly equipped laboratory has become thoroughly familiar with IF techniques, pediatric respiratory specimens can be screened for RSV by IF and only the IF-negative specimens need be inoculated into cell cultures for isolation of virus during the winter respiratory season.

Acute Disease↗

Clinical evaluation of an automated enzyme-linked fluorescent assay for the detection of chlamydial antigen in specimens from high-risk patients.

A fully automated enzyme-linked fluorescent assay (ELFA) on the Vitek Immunodiagnostic Assay System (VIDAS CHL) was evaluated for the detection of chlamydial antigen in specimens from symptomatic and asymptomatic high-risk patients. The results were compared with those from McCoy cell culture and Chlamydiazyme with a blocking assay. False-positive VIDAS specimens were centrifuged and the pellet stained with direct fluorescent antibody (DFA). Of the 158 urine specimens, 52 (33%) were infected by Chlamydia trachomatis. The sensitivity and specificity of the VIDAS when compared with cell culture, DFA, and Chlamydiazyme were 75% and 96%, respectively, for urine specimens while the predictive value of a positive (PVP) and a negative (PVN) were 91% and 88%, respectively. Of the 245 urethral swabs, 75 (31%) were considered positive. The sensitivity and specificity were 71% and 92%, respectively, and the PVP and PVN were 80% and 87%, respectively. The sensitivity and specificity on the 108 cervical swabs, 22 of which were positive, were 95% and 95%, respectively, and PVP and PVN were 88% and 99%, respectively. Compared with Chlamydiazyme, the VIDAS was more sensitive on specimens from female patients and urine specimens, but less sensitive on urethral specimens from male patients.

Adolescent↗

Chlamydia trachomatis confirmatory testing of PCR-positive genitourinary specimens using a second set of plasmid primers.

A plasmid-based PCR for the detection of Chlamydia trachomatis was evaluated with a confirmatory PCR employing a second set of plasmid primers. A total of 258 genitourinary specimens including 134 female endocervical and urethral specimens and 124 male urethral specimens were tested by culture, blocked EIA and PCR. Fifty-four specimens were positive by culture, 50 were positive by EIA and 71 were positive by PCR. Fourteen specimens that were PCR-positive but culture- and EIA-negative were confirmed positive by the confirmatory PCR. Two of the 187 specimens which were negative by culture and EIA were positive by PCR but failed to confirm with the second set of primers. Using an expanded gold standard of culture, blocked EIA and confirmed PCR, the overall sensitivities for culture, blocked EIA and confirmed PCR were 76.0% (54/71), 70.4% (50/71) and 100% (71/71) and the specificities were 100% (187/187), 100% (187/187), respectively. These results demonstrated that a confirmatory PCR was useful for sorting out discordant specimens and establishing the true specificity of PCR. Furthermore, these results demonstrate that PCR is more sensitive than culture and EIA and suggest that a confirmed PCR test should be included in the gold standard for the evaluation of new tests for diagnosing Chlamydia trachomatis infections.

Chlamydia Infections↗

How many sputum specimens are necessary to diagnose pulmonary tuberculosis?

The definitive diagnosis of pulmonary tuberculosis (PTB) relies on identifying or culturing Mycobacterium tuberculosis from respiratory specimens. National guidelines have recommended obtaining 3 sputum specimens from patients with suspected tuberculosis, but there has been little data on the number of specimens actually needed to support a diagnosis. We retrospectively reviewed all patients diagnosed with PTB at a public inner-city hospital and assessed the sensitivity of the acid-fast bacilli (AFB) smear and the number of smears needed to establish the diagnosis. Between January 1, 1997 and October 1, 2000, 425 patients were diagnosed with culture-proven PTB. Acid-fast bacilli (AFB) smears and cultures were performed on 951 respiratory specimens from 425 patients. The overall sensitivity of a positive AFB smear increased from 67% with 1 sputum collected to 71% and 72%, respectively, with the second and third specimens. The sensitivity of smears from 239 HIV-negative patients was 75%, 79%, and 80% with 1, 2, and 3 smears, respectively, collected compared with 57%, 61%, and 62%, respectively, for 142 HIV-positive patients. In summary, 2 respiratory specimens proved adequate in establishing a diagnosis of tuberculosis, and the third specimen added little additional diagnostic value.

Adolescent↗

Direct detection of Mycobacterium tuberculosis in clinical specimens using single-tube biotinylated nested polymerase chain reaction-enzyme linked immunoassay (PCR-ELISA).

A biotinylated single-tube nested polymerase chain reaction (PCR) assay with microwell hybridization assay (bPCR-ELISA) was developed for detection of Mycobacterium tuberculosis in clinical specimens. A total of 659 specimens (601 respiratory specimens and 58 nonrespiratory specimens) were collected for evaluation using three DNA amplification techniques: newly designed bPCR-ELISA, in-house single-tube nested PCR for IS6110 gene sequence (nPCR), and commercial automated assays, the Cobas Amplicor System from Roche Diagnostic Systems (aPCR). Sixty-four (9.7%) specimens were culture-positive for M. tuberculosis. Eleven (1.7%) specimens culture-positive for nontuberculosis mycobacteria were negative by all three PCR assays. The resolved performance of bPCR-ELISA, nPCR, and aPCR was found at sensitivities of 97%, 94%, and 97%, respectively. All three PCR assays exhibited a 100% specificity. In evaluation of bPCR-ELISA, a clear distinction between PCR-positive and PCR-negative specimens when an OD405 value of 0.6 was chosen as cut-off. With serial dilutions of M. tuberculosis H37Rv DNA, the detection limit of bPCR-ELISA was found to be 0.75 cfu per reaction at OD405 value of 0.6. Our developed bPCR-ELISA provides a highly sensitive and low-costing molecular diagnosis suitable for developing countries with high prevalence of tuberculosis.

Bacteriological Techniques↗

Transcript signatures of lymphocytic bronchitis in lung allograft biopsy specimens.

BACKGROUND: Rejection and obliterative bronchiolitis are barriers to sustained graft function in recipients of transplanted lungs. Early detection is hindered by inadequate tests and an incomplete understanding of the molecular events preceding or accompanying graft deterioration. METHODS: Hypothesizing that genes involved in immune responses and tissue remodeling produce biomarkers of rejection, we measured the expression of 192 selected genes in 72 sets of biopsy specimens from human lung allografts. Gene transcripts were quantified using a 2-step, multiplex, real-time polymerase chain reaction approach in endobronchial and transbronchial biopsy specimens from transplant recipients without acute infections undergoing routine surveillance bronchoscopy. RESULTS: Comparisons of histopathology in parallel biopsy specimens identified 6 genes correlating with rejection as manifested by lymphocytic bronchitis, a suspected harbinger of obliterative bronchiolitis. For example, beta2-defensin and collagenase transcripts in inflamed bronchi increased 37-fold and 163-fold, respectively. By contrast, these transcripts did not correlate with acute rejection in transbronchial specimens. Further, no correspondence was noted between histopathologic bronchitis and parenchymal rejection when endobronchial and transbronchial samples were obtained from the same patient. CONCLUSIONS: Our highly sensitive method permits quantitation of many gene transcripts simultaneously in small, bronchoscopically acquired biopsy specimens of allografts. Transcript signatures obtained by this approach suggest that airway and alveolar responses to rejection differ and that endobronchial biopsy specimens assess lymphocytic bronchitis and chronic rejection but are not proxies for transbronchial biopsy specimens. Further, they reveal changes in airway expression of the specific genes involved in host defense and remodeling and suggest that the measurement of transcripts correlating with lymphocytic bronchitis may be diagnostic adjuncts to histopathology.

Adult↗

A novel method for freezing and storing research tissue bank specimens.

Preserving small pieces of frozen tissue for possible future ancillary studies ("tumor banking") usually involves either placing a small piece of tissue in a cryovial and snap freezing it in liquid nitrogen or embedding and freezing the tissue in a block of cryopreservation medium, such as optimal cutting temperature (OCT) compound. The cryovial storage method leaves an irregularly shaped piece of tissue frozen to the side of the plastic vial, where it is exposed to air, subject to desiccation ("freezer burn"), and difficult to remove, but the vials are easy to store. The OCT method results in good morphological preservation, but yields a large awkwardly stored block from which it may be difficult to locate and recover small specimens. We have proposed a novel method of storing tissue bank specimens, the "capsule-freeze" method, which combines the advantages of OCT specimen preservation with those of cryovial specimen storage. Using this method, a tissue specimen is "snap-frozen" in OCT within a size "00" VCap pharmaceutical capsule, then the capsule is stored within a 1.5-mL cryovial. The specimen is harvested by simply cutting a slice out of the capsule and sealing the cut ends with OCT before their return to the freezer. The slice is then embedded en face within an OCT block before frozen sectioning. Morphological preservation is excellent, and the capsules are very easy to store. Occasional cracking that we found with the use of gelatin capsules is greatly diminished with the use of VCaps cellulose-walled capsules. The OCT can be easily removed by rinsing in cold 70% ethanol solutions. This method of tissue storage is ideal for the small specimens that are now commonly archived in these days of tissue sparing surgery.

Capsules↗

Finite element models predict cancellous apparent modulus when tissue modulus is scaled from specimen CT-attenuation.

High-resolution architecture-based finite element models are commonly used for characterizing the mechanical behavior of cancellous bone. The vast majority of studies use homogeneous material properties to model trabecular tissue. The objectives of this study were to demonstrate that inhomogeneous finite element models that account for microcomputed tomography-measured tissue modulus variability more accurately predict the apparent stiffness of cancellous bone than homogeneous models, and to examine the sensitivity of an inhomogeneous model to the degree of tissue property variability. We tested five different material cases in finite element models of ten cancellous cubes in simulated uniaxial compression. Three of these cases were inhomogeneous and two were homogeneous. Four of these cases were unique to each specimen, and the remaining case had the same tissue modulus for all specimens. Results from all simulations were compared with measured elastic moduli from previous experiments. Tissue modulus variability for the most accurate of the three inhomogeneous models was then artificially increased to simulate the effects of non-linear CT-attenuation-modulus relationships. Uniqueness of individual models was more critical for model accuracy than level of inhomogeneity. Both homogeneous and inhomogeneous models that were unique to each specimen had at least 8% greater explanatory power for apparent modulus than models that applied the same material properties to all specimens. The explanatory power for apparent modulus of models with a tissue modulus coefficient of variation (COV) range of 21-31% was 13% greater than homogeneous models (COV=0). The results of this study indicate that inhomogenous finite element models that have tissue moduli unique to each specimen more accurately predict the elastic behavior of cancellous cubic specimens than models that have common tissue moduli between all specimens.

Absorptiometry, Photon↗

Development and evaluation of a real-time RT-PCR assay on the LightCycler for the rapid detection of enterovirus in cerebrospinal fluid specimens.

BACKGROUND: Detection of enteroviral nucleic acid in cerebrospinal fluid (CSF) specimens has been demonstrated to improve the management of patients with aseptic meningitis. OBJECTIVE: To develop on the LightCycler (LC) instrument a real-time RT-PCR assay based on TaqMan technology for the detection of enteroviruses (EV) in cerebrospinal fluid (CSF) specimens. STUDY DESIGN: After evaluation of the analytical performances, seventy-four CSF samples collected prospectively from patients who have been suspected for a clinical diagnosis of meningitis were evaluated by two LC real-time RT-PCR assays and one conventional RT-PCR assay. RESULTS: Our assay detected all 30 different EV species tested, whereas no reactivity was observed with other neurotropic viruses. The analytical sensitivity of both LC RT-PCR real-time assays was 1 TCID50 for LC one-step and two-step RT-PCR assays. Results for LC one-step and LC two-step RT-PCR were compared to results of the conventional RT-PCR: of the 74 CSF specimens tested, 11 were positive and 56 were negative by all methods. Four other specimens were positive for EV by at least two of the methods (including the LC two-step RT-PCR and the conventional RT-PCR), two other CSF specimens were positive by the LC two-step RT-PCR assay only, and another one CSF specimen was positive by the LC one-step RT-PCR assay only. No CSF specimens were negative by the LC two-step RT-PCR assay and positive by the conventional RT-PCR assay. The sensitivity, specificity, positive and negative predictive values of both LC RT-PCR assays by using conventional RT-PCR as the "gold standard" were, respectively, 73.3, 98.3, 91.7, 93.5% for the LC one-step RT-PCR and 100, 96.6, 88.2, 100% for the LC two-step RT-PCR. There was substantial agreement between the three assays (k=0.80). CONCLUSIONS: The LC two-step RT-PCR assay is a rapid, sensitive and reliable method which can be routinely performed with CSF samples for diagnosis of EV infection and is an important improvement for optimal patient management.

Cerebrospinal Fluid↗

Fine-needle aspiration biopsy specimen with a predominance of Hürthle cells: a dilemma in the management of nodular thyroid disease.

BACKGROUND: A fine-needle aspiration biopsy (FNAB) specimen of a thyroid nodule with a predominance of Hürthle cells usually is indicative of a Hürthle cell neoplasm, but it also may occur with nonneoplastic disease. METHODS: A prospective nodular thyroid disease database was used to identify patients with a FNAB specimen consisting of a predominance of Hürthle cells. Clinical factors were investigated and FNAB specimens were examined in a blinded fashion by a single cytopathologist to determine if there were specific factors that could be used to distinguish nonneoplastic from neoplastic disease. RESULTS: Of the 738 patients with nodular thyroid disease, 622 had a FNAB specimen. The FNAB specimen was interpreted as consistent with a Hürthle cell neoplasm in 45 (7%) patients, 7 (16%) with carcinoma, 21 (47%) with adenoma, 12 (27%) with adenomatous hyperplasia, and 5 (11%) with thyroiditis. Extensive cellularity and absent colloid were associated with neoplastic disease (P < .05). No cytologic feature reliably excluded neoplastic disease (P > .05). No significant differences in age (x +/- SD) (51 +/- 17 vs 54 +/- 17 y), sex (female/male ratio, 6/1 vs 15/2), nodule size (3.9 +/- 1.9 vs 3.4 +/- 2.0 cm), weight of excised thyroid tissue (42 +/- 27 vs 33 +/- 30 g), or functional status of the thyroid gland was observed between patients with neoplastic (n = 28, 62%) versus nonneoplastic (n = 17, 38%) disease. CONCLUSIONS: Neoplastic disease accounts for two thirds of the pathology in patients with a predominance of Hürthle cells on FNAB specimen and neither clinical nor cytologic features reliably exclude Hürthle cell adenoma or carcinoma. As a result, thyroidectomy is recommended for all patients with a thyroid nodule and a predominance of Hürthle cells on FNAB specimen.

Adenoma, Oxyphilic↗

On specimen tilt for electron holography of semiconductor devices.

Electron holography can be successfully used for potential mapping on a nanometer scale. It relies on the fact that the phase of the electron wave is proportional to the electrostatic potential in the specimen. However, this proportionality is valid only in a kinematical condition, which is achieved by proper specimen orientation with respect to the electron beam. In this report, we examine experimentally in detail the specimen orientations of silicon devices that minimize dynamical diffraction. The tilt of the specimen from the edge-on position to a favorable orientation causes certain interfaces in the specimen to smear. We describe the smearing by a transfer function and compare it to wave transfer function of the microscope. The maximal specimen tilt alphamax that does not cause smearing greater than the resolution r of the microscope is alphamax = arctan(5.70r/t), where t is the specimen thickness.

Journal Article↗

Influence of endoscopic biopsy forceps characteristics on tissue specimens: results of a prospective randomized study.

BACKGROUND: A large variety of endoscopic biopsy forceps are commercially available. However, little is known regarding the influence of forceps characteristics such as disposability, size, shape, and presence of a needle on the adequacy of the specimens for histologic diagnosis. Our aim was to analyze in a prospective, randomized, pathologist-blinded study the performance of different biopsy forceps. METHODS: Twelve biopsy forceps were tested, 6 each at upper endoscopy and colonoscopy. Two biopsy specimens were obtained with each forceps, for a total of 12 specimens per patient. The tissue samples were examined for the following parameters: weight (mg), size (mm3), depth, crush artifact, sheering effect, and adequacy of the specimens for histologic information (0 = inadequate, 1 = suboptimal, and 2 = adequate). RESULTS: Fifty-five patients undergoing routine upper or lower gastrointestinal endoscopy were included in the study, and a total of 624 tissue samples were available for analysis. Overall, disposable forceps provided specimens of greater size and depth. At upper endoscopy, alligator-shaped forceps improved the depth of the sample as did the absence of a needle within the cup. These factors, however, had no impact on the specimens obtained at colonoscopy. When the adequacy of the specimens was assessed for histologic diagnosis, no significant difference was noted between any of the individual forceps, although collectively oval-shaped forceps were superior to alligator-shaped forceps at colonoscopy. CONCLUSIONS: The biopsy forceps currently available in the market are equally efficient in providing histologic diagnosis. The primary consideration when selecting an endoscopic biopsy forceps, therefore, should be the cost and ease of use and not any perceived advantage in performance.

Adult↗