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Evaluation of two commercially available nucleic acid hybridization assays for the detection and typing of human papillomavirus in clinical specimens.

Southern blot (Oncor, Gaithersburg, MD) and dot blot (Life Technologies, Gaithersburg, MD) nucleic acid hybridization assays were compared for their ability to detect and type human papillomavirus (HPV) DNA in 50 cervical swab specimens and 11 biopsy specimens. Overall agreement between the two methods was 78.7%. With the use of Southern blot analysis, HPV 6, 11, 16, or 18 was detected in 22 specimens, however, 4 were untypable because of abnormal or smeared band patterns. Dot blot analysis detected HPV 6/11, 16/18, or 31/33/35 in those same 22 specimens and in 9 additional specimens. Eight of the 13 specimens in which HPV was not detected or untypable by Southern blot contained type 31/33/35 by dot blot. Based on convenience of specimen collection and transport, ease of performance and the ability to detect HPV types 31, 33, and 35, the authors are currently using the dot blot assay for the detection and typing of HPV in clinical specimens.

Adolescent↗

Evaluation of patient-administered tampon specimens for Chlamydia trachomatis and Neisseria gonorrhoeae.

BACKGROUND: The patient-administered tampon specimen has proven to be an easy and sensitive method for the diagnosis of genital Chlamydia trachomatis and Neisseria gonorrhoeae infections in women by polymerase chain reaction (PCR). This method avoids the need for endocervical sampling and stringent criteria for transport. GOAL: To evaluate two commercial amplification systems for the detection of C trachomatis and N gonorrhoeae from tampon specimens. STUDY DESIGN: A group of 400 positive and negative tampon specimens tested by an in-house PCR method were selected from a pool of more than 2,000 previously collected tampons. Overall, 93 C trachomatis-positive and 77 N gonorrhoeae-positive specimens were evaluated. Each specimen was tested by Roche Cobas Amplicor and Abbott LCx (LCR), and results were compared to the in-house PCR method. RESULTS: Detection of C trachomatis by both assays was not significantly different from the in-house PCR assay. Fewer tampons were positive for N gonorrhoeae by LCR than either the in-house assay (P = 0.0001) or by Roche Amplicor (P = 0.01). However, tampon specimens tested by Roche Amplicor required DNA extraction to achieve comparative sensitivity. CONCLUSION: Both commercial assays can be applied to tampon-collected specimens for automated detection of sexually transmitted diseases. The detection of C trachomatis was similar to the in-house PCR test for both assays (P = 0.73, 0.68). Detection of N gonorrhoeae resulted in fewer positive tampon specimens when tested by ligase chain reaction than both Roche Amplicor and in-house PCR.

Chlamydia Infections↗

Use of pooled formalin-preserved fecal specimens to detect Giardia lamblia.

Three formalin-preserved fecal specimens from the same child attending a child-care center were pooled and compared with the three separate individual specimens by a single microscopic examination of concentration sediment for Giardia lamblia. The sensitivity of the pooled system was 100% when two or more individual specimens were positive and 88% when only one individual specimen was positive. The organism density in a single specimen was not a factor of whether the pool of specimens was positive or negative. Nearly half of the pools that contained positive specimens had only one of three specimens with positive results, reinforcing the need for multiple stool examinations when diagnosing G. lamblia infections.

Animals↗

Evaluation of Amplicor PCR for direct detection of Mycobacterium tuberculosis from sputum specimens.

We evaluated the Amplicor PCR assay (Roche Molecular Systems, Branchburg, N.J.) for direct detection of Mycobacterium tuberculosis in sputum. A total of 532 specimens from 270 patients were decontaminated and stored at 4 or -75 degrees C until assayed by PCR. This assay used three-step sample preparation, biotinylated primer pairs, AmpErase, and a microtiter format for amplicon capture and detection. Amplicor PCR results were compared with clinical history, culture from a Lowenstein-Jensen slant, and results from the BACTEC TB-460 system. Eighty-seven cultures from 15 patients grew M. tuberculosis; of these, 83 (95%) were positive with the Amplicor PCR test. The false negatives were most likely due to sample variation and inhibitors. Of the 445 specimens from which M. tuberculosis was not isolated, 428 (96%) were negative with the Amplicor PCR test. Of the 17 M. tuberculosis culture-negative, Amplicor-positive specimens, 15 were reclassified as true positives because previous cultures grew M. tuberculosis. Of the 445 specimens which did not grow M. tuberculosis, Mycobacterium spp. other than M. tuberculosis were isolated from 150 specimens. Three of these 150 specimens were Amplicor positive; two were from a patient with a history of tuberculosis, and one specimen gave a false-positive result. We do not feel that this represents cross-reactivity, because repeated Amplicor testing of the isolate gave negative results. The microtiter plate has 96 wells. Allowing for six controls, 90 decontaminated specimens can be tested by one technologist in 7.5 h. This PCR assay took 7.5 h to complete and is a sensitive and specific, rapid method for the direct detection of M. tuberculosis from sputum.

Case Management↗

Mailed, home-obtained urine specimens: a reliable screening approach for detecting asymptomatic Chlamydia trachomatis infections.

The use of mailed, home-obtained urine specimens could facilitate screening programs for the detection of asymptomatic Chlamydia trachomatis infections. Since transport time could have an adverse effect on the sensitivity of C. trachomatis detection by PCR, the influence of DNA degradation on amplification was monitored over the course of 1 week. Therefore, urine specimens were aliquoted on the day of collection or arrival. Two groups of urine specimens were investigated. Group I contains first-void C. trachomatis-positive and -negative urine samples. DNA degradation was monitored in group I samples for 7 days at room temperature (RT) and at 4 degrees C by amplifying different lengths of the human beta-globin gene and the C. trachomatis plasmid target. DNA degradation was observed only for the larger human beta-globin fragments at days 5 to 7 at RT. In contrast, at 4 degrees C all targets could be amplified. Urine specimens were also frozen and thawed before aliquoting to mimic freezing during transport. This resulted in a lower sensitivity for the detection of C. trachomatis after thawing and 3 to 4 days at RT. In addition, mailed, home-obtained C. trachomatis-positive urine specimens (group II) were analyzed for 7 days after arrival by two commercially available C. trachomatis detection systems (PCR and ligase chain reaction [LCR]). The C. trachomatis plasmid target in mailed, home-obtained urine specimens could be amplified by both PCR and LCR after 1 week of storage and/or transport at RT. In conclusion, our findings indicate that mailed, home-obtained urine specimens are suitable for the sensitive detection of asymptomatic C. trachomatis infections by amplification methods, even if the transport time is up to 1 week at RT. These findings support the feasibility and validity of screening programs based on mailed, home-obtained urine specimens. Larger studies should be initiated to confirm our results.

Base Sequence↗

Laboratory assessment of the status of Her-2/neu protein and oncogene in breast cancer specimens: comparison of immunohistochemistry assay with fluorescence in situ hybridisation assays.

AIM: To evaluate the clinical usefulness of three commercially available assays for Her-2/neu oncogene and protein measurements. The Her-2/neu protein is overexpressed, mostly as a result of gene amplification, in 20-30% of human breast cancers, and has been shown to have prognostic and predictive value for treatment with chemotherapy or the new monoclonal antibody, Herceptin. METHODS: An immunohistochemistry (IHC) assay using the Dako polyclonal antibody A0485, which measures the Her-2/neu protein, was compared with two new Food and Drug Administration (FDA) approved fluorescence in situ hybridisation (FISH) assays--INFORM and PathVysion, in a cohort of 52 formalin fixed, paraffin wax embedded breast tissues. These tissues were selected randomly from 84 consecutive infiltrating breast cancer specimens, which were first stratified according to the Her-2/neu protein levels as measured by IHC. RESULTS: The two FISH assays achieved a 98% concordance rate: 14 specimens (27%) showed Her-2/neu gene amplification and 37 specimens (71%) showed no Her-2/neu gene amplification. The PathVysion assay had certain advantages over the INFORM assay. In contrast, the IHC assay detected Her-2/neu overexpression in a high percentage of cases, including 13 high positive specimens (25%) and 13 medium positive specimens (25%). Although 10 of these 13 IHC high positive specimens showed gene amplification by FISH, nine of 13 IHC medium positive specimens showed no gene amplification. Statistical analyses showed that the differences between IHC and FISH assays were primarily in the specimens with medium positive IHC, but negative FISH results. CONCLUSIONS: Because of the increasing importance of the Her-2/neu oncogene and oncoprotein in the clinical management of patients with breast cancer, the accurate and consistent evaluation of Her-2/neu status is crucial. This study suggests that the best approach is to combine both IHC and FISH assays; that is, to use the IHC assay as a triage step, followed by the PathVysion FISH assay to analyse the IHC medium and high positive cases.

Biomarkers, Tumor↗

Preservation of brain tumor specimens for drug sensitivity testing.

To examine the feasibility of shipment of brain tumor specimens to a central laboratory for drug sensitivity testing, an experimental gliosarcoma (9L) was grown subcutaneously in rats, harvested, stored, and disaggregated. Growth of the disaggregated tumor cells in monolayer culture was evaluated after storage for various times at 2 to 6 degrees C and 37 degrees C in saline and minimum essential medium. Growth potential was maintained for 24 hours when tumor specimens were stored under refrigerated conditions and was best maintained when specimens were stored in saline. Specimens stored at 37 degrees C grew best when stored in minimum essential medium, but growth potential was lost after 12 hours unless the specimens were refrigerated. Shipment of tumor specimens to a central laboratory for drug sensitivity testing appears to be feasible, since under most circumstances, specimens should reach the laboratory for processing within 24 hours of their removal. Storage and transport of specimens in saline on wet ice appears to be optimal.

Antineoplastic Agents↗

Outpatient phlebotomy success and reasons for specimen rejection.

OBJECTIVES: To determine the rate with which blood collection is successful on the initial phlebotomy encounter, the rate with which laboratory personnel judge specimens unsuitable for analysis, and the practice characteristics associated with fewer unsuccessful collections and fewer rejected specimens. DESIGN: Clinical laboratories participating in the College of American Pathologists Q-Probes laboratory improvement program prospectively characterized the outcome of outpatient phlebotomies for 3 months or until 20 unsuccessful phlebotomy encounters occurred. By questionnaire, participants provided information about test ordering, patient preparation, and specimen collection. SETTING AND PARTICIPANTS: Institutions in the United States (n = 202), Canada (n = 4), Australia (n = 3), and South Korea (n = 1). MAIN OUTCOME MEASURES: Percentage of successful encounters and percentage of unsuitable specimens. RESULTS: Of 833289 encounters, 829723 were successful. Phlebotomies were unsuccessful because patients were not fasting as directed (32.2%), phlebotomy orders were missing information (22.5%), patients specimens were difficult to draw (13.0%), patients left the collection area before specimens were collected (11.8%), patients were improperly prepared for reasons other than fasting (6.3%), patients presented at the wrong time (3.1%), or for other reasons (11.8%). Only 2153 specimens (0.3%) were unsuitable; these samples were hemolyzed (18.1%), of insufficient quantity (16.0%), clotted (13.4%), lost or not received in the laboratory (11.5%), inadequately labeled (5.8%), at variance with previous or expected results (4.8%), or unacceptable for other reasons (31.1%). Facilities staffed by laboratory-administered phlebotomists reported higher success rates than facilities staffed by nonlaboratory-administered phlebotomists (P =.002). CONCLUSIONS: Most outpatient phlebotomy encounters are successful and result in specimens suitable for laboratory analysis.

Australia↗

[Evaluation of the new detectable method of the specimen mix-up in a microbiological examination].

To date, it has been difficult to detect a mix-up of specimens for microbiological examinations. We developed and evaluated a computer program that detects specimen mix-up during the examinations. Once some bacterial species such as Pseudomonas aeruginosa and MRSA are detected in an examination, it is not easy for these bacteria to disappear on subsequent examinations. It is possible to speculate that there has been a specimen mix-up when there are differences in the existence of bacterial species between the results of the past and current examinations. In this study, 1379 false mix-up specimens were created and evaluated for the detection of specimen mix-ups using this method and 417 of the 1379 specimens were detected as mix-ups (sensitivity; 30.2%). Furthermore, when 1486 real specimens were evaluated for the detection of mix-ups, 114 samples were judged to have been mixed-up. Three of these samples were mixed up during the examinations (specificity; 92.5%). These findings suggest that this method is useful for the detection of specimen mix-up during microbiological examinations.

Bacteriological Techniques↗

DNA probes for the detection of mycoplasmas in genital specimens.

The utility of whole-genomic DNA probes for the detection of infections by genital mycoplasmas was investigated in 220 men attending a sexually transmitted diseases clinic. In 144 patients, probe results were compared with quantitative culture results. The prevalence of Mycoplasma hominis was 11% by culture, whereas the prevalence of ureaplasmas was 38%. The M. hominis DNA probe detected 9 of 16 M. hominis culture-positive specimens and 2 of 128 culture-negative specimens. The Ureaplasma urealyticum DNA probe detected 36 of 57 U. urealyticum culture-positive specimens and 18 of 87 culture-negative specimens. Most of the probe-negative culture-positive specimens had colony counts of less than 10(3) organisms/ml of specimen. The DNA probe does not require viable organisms, and the probe-positive, culture-negative specimens suggest that false-negative cultures occurred, perhaps due to specimen handling or insensitivity of culture methods for some strains of mycoplasmas.

Bacteriological Techniques↗

Further studies of specimen volume changes during processing for SEM: including some plant tissue.

The dimensions of specimens undergoing preparation for examination in the SEM were measured throughout the preparative sequence or at various important stages. The tissues studied included 15-day mouse embryo limbs (MEL), small blocks of adult mouse liver and brain, and potato tuber. The animal tissues were fixed in 3% glutaraldehyde (GA) in 0.15M cacodylate buffer, and the potatoe tissue in 2% GA in water. The effects of various secondary fixation and other treatments were investigated. The results show that lithium salts cause a reduction in the shrinkage of MEL in 100% ethanol but this effect was not significant in the other tissues investigated, and did not persist in specimens stored after critical point drying (CPD). All CPD specimens were shrunken. However postosmication and treatment with uranyl acetate (UAc) and cetyl pyridinium chloride (CPC) all reduced specimen shrinkage in 100% ethanol and after critical point drying. The volume gains with Os + UAc and Os + CPC are both very significant, but it was found that these larger specimens shrank more on storage. Thus rapid examination in the SEM is recommended. Ethanol and Freon 113 were compared as intermediate fluids and it was found that ethanol-CO2 critical point dried specimens shrank more before and after CPD than Freon 113-CO2 specimens. The latter technique is, therefore, to be recommended. Potato tissue shrinks in 30% ethanol, whereas animal tissues all swell in this concentration. The potato tissue also shrank very litte on critical point drying in contrast to the animal tissue specimens.

Animals↗

Electron irradiation effects in the EPMA quantitation of organic specimens.

Electron irradiation of organic specimens adversely affects the validity and reliability of quantitative microanalytical results through the loss of light element constituents of the specimen. Characteristic and continuum x-ray count rates will vary for the duration of the irradiation effects, which require electron doses of 10(-11) to 10(-8) C/micrometer 2. The magnitude of these variations is dependent upon the light element content, thickness and temperature of the specimen and upon the operating conditions used during analysis. The initial count rate at the onset of the electron beam is the most valid count rate to be used in the quantitation of organic specimens, but it can only be accurately determined when the analyzed element exists in high concentration within the specimen or when the beam is grossly defocused. Low temperature analysis will prolong the duration of the mass loss and decrease its magnitude, but erratic count rates, specimen charging due to condensation and inconsistent mass losses have been observed for different specimens. The physics and chemistry of the electron beam-specimen interactions impose limitations upon the potential accuracy in biological microanalysis whic, if ignored, can result in large errors in quantitative projections.

Biology↗

QAU verification: Ricerca's approach to EPA requirements for specimen disposition.

The U.S. Environmental Protection Agency (EPA) Federal Insecticide. Fungicide and Rodenticide Act and Toxic Substance Control Act Standards issued in 1989 presented a new task for the Quality Assurance Unit (QAU). Sections .190 and .195 of these Good Laboratory Practice (GLP) Standards require "quality assurance verification" prior to the disposal of certain specimens. These include "specimens from mutagenicity tests, specimens of soil, water, and plants, and wet specimens of blood, urine, feces, and biological fluids." QAU involvement in the specimen disposition process was a new concept. Other GLP Standards required specimen retention only as long as the quality of the preparation afforded evaluation: the QAU was not included in the procedure. The QAU verification step was included by the EPA to ensure that disposal of the prescribed specimens would not compromise the integrity of the study. A procedure to address verification of specimen disposition as required by the EPA GLP Standards has been implemented by Ricerca's QAU. This presentation provides a description of this approach to QAU verification.

Documentation↗

Monitoring percent free PSA in serial specimens: improvement of test specificity, early detection, and identification of occult tumors.

We have measured the serum concentration of prostate specific antigen (tPSA) and determined the percent free PSA (% fPSA) in serial specimens from 64 patients with prostate cancer, 35 patients with benign prostate hyperplasia (BPH), and 3 patients with prostitis. We found that the % fPSAs were not a constant for individual patients during the course of the disease. When we compared only the % fPSA of the first specimen of serial specimens from individual patients, who were largely untreated, 37% of BPH specimens were above 22%, whereas only 1.6% cancer samples were above that value. We also found that 67% of cancer specimens and 14% of BPH samples were below 8%, respectively. Although % fPSA distribution pattern remained similar between two types of specimens, less differentiation was found between BPH and prostate cancer in random specimens compared to the study using first specimens of an individual patient's serial samples. Percent fPSA apparently are affected by treatment. However, the most important benefit for the determination of % fPSA appears to be the sensitivity of % fPSA to identify occult tumors when the tPSAs were in the normal concentration range. Determination of % fPSA also seems to improve the specificity of tPSA, not only during screening for the differentiation between BPH and prostate cancer, but also during monitoring of treatment and recurrence.

Humans↗

Estrogen and progesterone receptor assays on breast carcinoma from mastectomy specimens.

To determine whether autolytic loss of estrogen (ER) and progesterone (PgR) receptors might affect results of assays performed on primary carcinoma samples taken from fresh mastectomy specimens rather than from biopsy specimens, a group of 71 cases were examined, in which both types of samples were assayed. The comparisons showed a small significant reduction in the proportion of positive ER assay results in mastectomy-primary specimens compared with biopsy specimens, although the frequency of high-binding ER results in the mastectomy-primary specimens was not reduced. The number of positive PgR assays on mastectomy-primary specimens was not reduced in comparison with biopsy specimens. Correlation coefficients for ER values in the paired data were r = 0.836 for biopsy versus mastectomy, and r = 0.795 for primary tumor versus nodal metastasis. For PgR biopsy versus mastectomy r = 0.664, and for PgR primary versus node r = 0.352. The mean quantitative ER and PgR values were significantly higher in axillary lymph nodal metastases than in primary carcinomas, and the higher nodal receptor levels were explained in part by significantly higher tumor cellularity. It is concluded that while loss of ER sufficient to cause a false-negative interpretation may possibly occur in a few cases during the course of mastectomy, as a general rule, significant losses do not occur. The performance of assays on axillary metastases in mastectomy specimens is advantageous because of their high cellularity.

Axilla↗

Androgen receptors in biopsy specimens of prostate adenocarcinoma. Heterogeneity of distribution and relation to prognostic significance of receptor measurements for survival of advanced cancer patients.

Needle biopsy specimens of primary adenocarcinoma and surgical specimens of carcinomatous nodal tissue were obtained from previously untreated clinical D stage prostatic adenocarcinoma patients. Assessment of the relation between specimen androgen receptor site content and survival using either scatterplots or Kaplan-Meier analyses showed specimen receptor content was a poor prognostic P greater than 0.1, of survival subsequent to orchiectomy or diethylstilbestrol (DES) therapy. The possibility that heterogeneity of specimen androgen receptor site content contributed to this finding was evaluated by comparing receptor content of multiple small or large tissue specimens from the same prostate gland of patients with benign prostatic hyperplasia or nonmetastatic prostatic cancer. This evaluation showed significant microheterogeneity of human prostate androgen receptor site content which was substantially masked in large tissue specimens. We conclude that microheterogeneity of human prostate androgen receptor site content compromises the use of biopsy specimen androgen receptor measurements as a prognostic of patient survival subsequent to initiation of hormonal therapy.

Adenocarcinoma↗

"Atypical" cells in fine-needle aspiration biopsy specimens of benign thyroid cysts.

BACKGROUND: Cystic lesions of the thyroid are common. Most are benign nodules with degenerative changes in a multinodular goiter. Fine-needle aspiration biopsy (FNAB) specimens from these cystic nodules usually are easily interpreted as benign. However, occasionally, cells with atypical features are encountered, increasing the possibility of a cystic malignant neoplasm. To the authors' knowledge, the microscopic features of these benign cells, presumed to be of cyst-lining origin, have not been well described to date. To refine the description of their morphologic features, with the belief that better recognition will avoid unnecessary surgery, the authors examined the cytologic and corresponding histologic features of thyroid cysts with "atypical" cells. METHODS: A total of 149 FNAB specimens from thyroid cysts containing atypical cells were identified. Seventy-five specimens with subsequent histologic correlation showing a benign cystic thyroid nodule were selected for study. The cytologic features of the atypical cyst-lining cells were reviewed and correlations were made with histologic, immunohistochemical, and clinical features. In addition, 12 FNAB specimens of histologically proven cystic papillary carcinoma diagnosed as atypical were reviewed for comparison. RESULTS: The majority of specimens (94%) were diagnosed cytologically as atypical thyroid cysts. However, in 29% of these specimens, a papillary or Hurthle cell neoplasm could not be excluded. The cytologic features of the atypical cells most often resembled classic reparative epithelial cells consistent with a cyst-lining origin. The most common features were cohesive flat sheets (84%), distinct cell borders (96%), nuclear enlargement (92%), nuclear grooves (79%), dense granular cytoplasm (79%), small distinct nucleoli (85%), fine chromatin (87%), and elongate to spindled cytomorphology (57%). In contrast to the atypical cells from benign cysts, cystic papillary carcinomas lacked the repair-like spindled cytomorphology, and showed nuclear crowding (100%), as well as papillary microarchitecture (50%), and rare intranuclear pseudoinclusions (42%). Histologically, the atypical cells in aspirate specimens corresponded to cyst-lining cells, which exhibited a spectrum ranging from flattened elongate cells with eosinophilic cytoplasm to more polygonal cells with abundant eosinophilic cytoplasm, enlarged irregular nuclei with pale chromatin, prominent nuclear grooves, and distinct nucleoli. Immunohistochemical staining of a subset of the resected thyroid cysts showed that the cyst-lining cells were positive for keratin and thyroglobulin, consistent with thyroid follicular cells. CONCLUSIONS: Atypical cyst-lining cells were found to have characteristic features (e.g., distinct cell borders, elongated shape, eosinophilic cytoplasm, and distinct nucleoli) and lacked nuclear crowding, intranuclear pseudoinclusions, and papillary architecture that, in many specimens, allowed them to be recognized as benign. The authors recommended that the subset of cells with the characteristic features described in the current study be reported as "consistent with benign cyst lining cells".

Biopsy, Fine-Needle↗

Cytophotometric analysis of corresponding cytological and histological cervical intraepithelial neoplasia grade III specimens.

Cytophotometric analysis of cervical intraepithelial neoplasia grade III (CIN III) was performed in 22 cytological smears (CS) and in 22 corresponding cytospin specimens retrieved from selected areas of paraffin-embedded tissues (PEC). The average time interval between cytological and histological diagnosis was 6 weeks. CIN III nuclei in CS and PEC specimen were Thionin-Feulgen stained and digitized. Beside the visual classification of DNA ploidy patterns, the 2.5c and 5c exceeding rates and the specimen mean and standard deviation values of 21 photometric features were also analyzed. It was shown that, although there was a significant correlation between DNA ploidy patterns in corresponding PEC and CS specimen, the DNA patterns were dissimilar in eight of 22 cases. The DNA index, as represented by 2.5c and 5c exceeding rates, was significantly higher in the CS specimen. High-resolution cytophotometric analysis of cell nuclei in CS and PEC specimens showed significant differences for a large number of nuclear photometric features. These findings can possibly be explained by differences in selection of CIN III cells from CS and PEC specimens and by differences between fixation procedures as used for the two techniques. It was concluded that cytophotometric data of CS and PEC specimens representing CIN III lesions should not be regarded as interchangeable.

Cytophotometry↗