Reasons For False-negative Results Research Articles

Artificial intelligence in glaucoma detection using color fundus photographs.

To explore the potential of artificial intelligence (AI) for glaucoma detection using deep learning algorithm and evaluate its accuracy for image classification of glaucomatous optic neuropathy (GON) from color fundus photographs. A total of 1375 color fundus photographs, 735 normal optic nerve head and 640 GON, were uploaded on the AI software for training, validation, and testing using deep learning model, which is based on Residual Network (Res Net) 50V2. For initial training and validation, 400 fundus images (200 normal and 200 GON) were uploaded and for the final training and testing 975 (535 normal and 440 GON) were uploaded later. Accuracy, sensitivity, and specificity were used to evaluate the image classification performance of the algorithm. Also, positive predictive value, negative predictive value, positive likelihood ratio, and negative likelihood ratio were calculated. The model used in the study showed an image classification accuracy of 81.3%, sensitivity of 83%, and specificity of 80% for the detection of GON. The false-negative grading was 17% and false-positive grading was 20% for the image classification of GON. Coexistence of glaucoma in patients with high myopia, early glaucoma in a small disc, and software misclassification of GON were the reasons for false-negative results. Physiological large cupping in a large disc, myopic or titled disc, and software misclassification of normal optic disc were the reasons for false-positive results. The model employed in this study achieved a good accuracy, and hence has a good potential in detection of GON using color fundus photographs.

Application Value of Ultrasound-Guided Fine Needle Aspiration Cytological Examination and Ultrasound Evaluation

In this paper, chronic inflammatory cell infiltration was seen during this period. The reason for the false-positive result may be the atypical proliferation of cells caused by chronic inflammatory stimulation with malignant cytology. 12 cases of false negative results, including 2 cases of type I nodules, 10 cases of type III nodules, postoperative histology and pathology were papillary carcinoma, there are many reasons for false negative results, including the size of the nodules, the puncture operator, the technical level, internal tissue composition and properties of the nodules all affect the accuracy of puncture and cytopathology results. The satisfaction of puncture specimens of each group was calculated and compared. For thyroid nodules with the maximum diameter ≤5 mm, the satisfaction rate of puncture specimens was 87.2%, and the diagnostic sensitivity and accuracy of ultras guided fine needle aspiration biopsy were relatively low. Cytological results were in poor agreement with postoperative histological and pathological results, so it was recommended to take active follow-up for thyroid nodules with the maximum diameter ≤5 mm to reduce unnecessary puncture. The overall puncture success rate is relatively high, ≤10 mm low-echo nodules or even ≤5 mm nodules can be cytological puncture, especially the kind of close to the capsule, trachea and recurrent laryngeal nerve nodules, for the largest diameter of >5 mm, the satisfaction rate of puncture specimens is relatively high is 93.2%. Moreover, the sensitivity and accuracy of ultrasound guided fine needle aspiration biopsy were relatively high, and the cytological results were in good agreement with the postoperative histological and pathological results. Therefore, it was feasible to confirm the diagnosis of suspected malignant nodules by ultrasound. The diagnostic in each group and the consistency between cytological diagnosis and histological diagnosis were analyzed by comparing the histological and pathological results after surgery. Ultrasound-guided fine needle aspiration biopsy can be used more effectively in clinical practice.

Validation of the Response Speed Rule to Predict the Efficacy of a Therapeutic Regimen in Myeloma

BACKGROUND Current treatment choices are based on generalized outcome data from clinical trials, but not all MM patients respond the same and a considerable percentage of them do not achieve a desirable treatment endpoint. Contradictory results based on the rate of descent of the monoclonal component (M-component) to predict long-term outcome have been published, but no to identify patients with insufficient response to a therapeutic regimen. The ability to identify treatment early on resistance could accelerate the introduction of another (and more effective) line of therapy. AIM Develop and validate a rule based on the rate of descent of the M-component, to predict the probability of disease resistance to reach a complete remission (CR) in MM patients. METHODS Three studies were conducted: exploratory, confirmatory, and clinical validation. A total of 87 patients treated between July 2014 and September 2018, were included for the first two. Patients who were unable to complete the planned treatment due to toxicity or comorbidities, and those treated for palliative purposes only, were excluded. A therapeutic regimen was considered to be effective if CR was achieved with it. Conversely, a therapeutic regimen was considered to be ineffective if disease progression was observed during treatment, or if CR was not achieved with it. The percentage of the daily decrease in the M-component achieved by each treatment cycle is calculated dividing the percentage of the decrease during the cycle by the number of days elapsed (n.nn% /day). Timing between measurement of the M-component do not differed more than 15% from the scheduled cycle time. In the exploratory study using the receiver operating characteristic (ROC) curve through SPSS v24, the ability to discriminate between effective and ineffective therapeutic regimen was investigated in 99 cycles, to identify the optimal cutoff, and to desing a rule; followed by a confirmatory study of the rule in 52 cycles different from those of the first study. A third clinical validation study was carried out with 62 patients, 31 with treatment guided by response speed rule (RSR-guided) and 31 not (unguided). RESULTS In the exploratory study it was observed that the area under the ROC curve was 0.971 (CI 95%: 0.93 - 1.00) (p<0.001) to predict ineffective therapeutic regimen. The optimal threshold was 1.405% /day. The test was considered positive for therapeutic ineffectiveness if ≤1,40% /day. Sensitivity 95.0% (CI 95%: 88.0 - 99.0), specificity 94.7% (CI 95%: 74.0 - 99.8). It was estimated that the false positive results arose due to the occasional lack of adherence of the patient to oral treatment. Also, we have observed that the first therapeutic cycle usually produces a greater decrease in the M component than the successive cycles, and this was the reason for false negative results. Thus, the Response Speed Rule (RSR) was defined as a descent of ≤ 1.40% /day in two successive cycles, and > 1.40% /day in a first cycle it does not indicate efficacy. In the confirmatory study it was observed for the RSR a sensitivity 100% (CI 95%: 92 - 100), specificity 100% (CI 95%: 66 - 100), reliability 100% (CI 95%: 93 - 100). In the clinical validation study the most common treatments in 1st, 2nd, and 3rd line were VBCMP/VBAD, VCD, and KRd for candidates, and VCD, Rd, DRd or KRd in non-candidates respectively. No significant differences were observed in the type of treatment used between RSR-guided and unguided patients. The median (months) to reach the CR in the RSR-guided patients was lower (8.5 vs 12.1 months; p = 0.003). RS-guided patients need fewer cycles to achieve CR (5.29 vs. 10.84; p = 0.002). The CR rate at 18 months was 94.3% (CI 95%: 84.1 - 100) and 74.2% (CI 95%: 58.4 - 90.0) for RSR-guided and unguided patients respectively, although in unguided patients the rate rose to 96.8% (CI 95%: 90.4 - 100.0) continuing treatment until 30 months. No significant differences were found in the number of lines for CR (1.96 vs 2.42; p = 0.054). CONCLUSIONS The Response Speed Rule (RSR), defined as a speed of M-component descent of ≤ 1.40% /day in two successive cycles, predicted with great accuracy the current ineffectiveness of a therapeutic regimen to deliver CR. During the clinical validation of this cutoff, it was shown that CR is achieved in less time and with fewer cycles using RSR. This is a simple metric that can be used broadly and accelerate the introduction of another (and more effective) line of therapy. Figure Disclosures No relevant conflicts of interest to declare.

Preoperative Axillary Ultrasound versus Sentinel Lymph Node Biopsy in Patients with Early Breast Cancer.

Background and objectives: With improved diagnostic means of early breast cancer, the percentage of cases with metastasis in axillary lymph nodes has decreased from 50–75% to 15–30%. Lymphadenectomy and sentinel lymph node biopsy are not treatment procedures, as they aim at axillary nodal staging in breast cancer. Being surgical interventions, they can lead to various complications. Therefore, recently much attention has been paid to the identification of non-invasive methods for axillary nodal staging. In many countries, ultrasound is a first-line method to evaluate axillary lymph node status. The aim of this study was to evaluate the prognostic value of ultrasound in detecting intact axillary lymph nodes and to assess the accuracy of ultrasound in detecting a heavy nodal disease burden. The additional objective was to evaluate patients’ and tumor characteristics leading to false-negative results. Materials and Methods: A total of 227 women with newly diagnosed pT1 breast cancer were included to this prospective study conducted at the Breast Surgery Unit, Clinic of Surgery, Hospital of Lithuanian University of Health Sciences Kauno Klinikos, between May 1, 2016, and May 31, 2018. All patients underwent preoperative axillary ultrasound examination. Ultrasound data were compared with the results of histological examination. The accuracy and true-negative rate of ultrasound were calculated. The reasons of false-negative results were analyzed. Results: Of the 189 patients who had normally appearing axillary lymph nodes on preoperative ultrasound (PAUS-negative), 173 (91.5%) patients were also confirmed to have intact axillary lymph nodes (node-negative) by histological examination after surgery. The accuracy and the negative predictive value of ultrasound examination were 84.1% and 91.5%, respectively. In ≥3 node-positive cases, the accuracy and the negative predictive value increased to 88.7% and 98.3%, respectively. In total, false-negative results were found in 8.5% of the cases (n = 16); in the PAUS-negative group, false-negative results were recorded only in 1.6% of the cases (n = 3). The results of PAUS and pathological examination differed significantly between patients without and with lymphovascular invasion (LV0 vs. LV1, p < 0.001) as well as those showing no human epidermal growth factor receptor 2 (HER2) expression and patients with weakly or strongly expressed HER2 (HER2(0) vs. HER2(1), p = 0.024). Paired comparisons revealed that the true-negative rate was significantly different between the LV0 and LV1 groups (91% vs. 66.7%, p < 0.05), and the false-negative rate was statistically significant different between the HER2(0) and HER2(1) groups (10.5% vs. 1.2%, p < 0.05). Evaluation of other characteristics showed both the groups to be homogenous. Conclusions: Negative axillary ultrasound excluded axillary metastatic disease in 91.5% of the patients. PAUS had an accuracy of 88.7% in detecting a heavy nodal disease burden. With the absence of lymphovascular invasion (LV0), we can rely on PAUS examination that axillary lymph nodes are intact (PAUS-negative), and this patients’ group could avoid sentinel lymph node biopsy. Patients without HER2 expression are at a greater likelihood of false-negative results; therefore, the findings of ultrasound that axillary lymph nodes are intact (PAUS-negative results) should be interpreted with caution.

Evaluation of phototoxic and cytotoxic potential of TiO2 nanosheets in a 3D reconstructed human skin model

Despite a continuous increase of commercial products containing nanoparticles, only few materials are currently used in such large amounts, forms and sizes as titanium dioxide - TiO2. Besides its use in cosmetics, food industry and biomedicine, TiO2 nanoparticles (NPs) are also used as highly efficient photocatalysts due to their unique ability to convert complex organic materials to carbon dioxide, water and simple mineral acids via complex radical and electron transfer reactions. The main objective of the current project was to assess the potential dermal effect (phototoxicity and skin toxicity) of TiO2 nanosheets (TIG-800) synthesized from the lyophilized aqueous colloids of peroxo-titanic acid by high-temperature treatment. The effect was examined on reconstructed human skin model EpiDerm in the test pre-validated by ECVAM and adopted into the ICH S10 guidelines for the preclinical photo-safety assessment of drugs. In addition, two further commercial samples of nanocrystalline TiO2, namely Aeroxide P25 and Eusolex T-2000, and six benchmark materials from pre-validation studies were tested. None of the TiO2 NPs tested in the study caused acute phototoxicity or cytotoxicity in the reconstructed 3D tissues up to the highest concentration tested. The prediction of photo-irritation potency for the benchmark chemicals was comparable to previous studies. For some of the tested materials, we identified reasons for false negative results or variability in previously published datasets by improved dosing, conditions of irradiation, and choice of suitable solvents. The method proved its suitability for photo-irritation assessment of topically applied materials.

Detection of central venous catheter-related bloodstream infections in haematooncological patients.

Catheter-related bloodstream infections (CRBSIs) are currently detected in patients with clinically suspicion. The aim of our study was to evaluate whether CRBSIs could be anticipated and detected in a subclinical stage by peptide nucleic acid fluorescence in situ hybridization (PNA FISH) using universal hybridization probes or acridine orange leucocyte cytospin (AOLC) tests in haematooncological patients with central venous catheters (CVCs) in situ. Peptide nucleic acid fluorescence in situ hybridization and AOLC tests using blood samples from one CVC lumen/port chamber in haematooncological patients were continuously performed. These results were compared to those obtained from routinely performed CRBSI diagnostic tests. One hundred and eighty-two patients with 342 catheter periods were investigated. Seventeen CRBSI cases were detected in 6466 CVC days by routine measures resulting in a CRBSI rate of 2.6/1000 catheter days. Two of 17 showed positive PNA FISH tests, and five positive AOLC test results before the diagnosis were established with routine measures. The screening revealed further seven patients with positive universal PNA FISH tests and 10 positive AOLC tests without symptoms indicative for infection and were therefore considered not to have CRBSI. Sampling of only one CVC lumen/port chamber screening for CRBSI in haematooncological patients seems not to be a useful tool for anticipative diagnosis of CRBSI. Reasons for false-negative results might include origin of CRBSIs from the other CVC lumina not sampled for screening, and false-positive results might origin from catheter colonization without subsequent spread of micro-organisms into the peripheral bloodstream.

Editorial Comment from Dr Borkowska to Recurrence and progression in patients with non‐muscle invasive bladder cancer: Prognostic models including multicolor fluorescence in situ hybridization molecular grading

In this issue of the journal, Lodde et al. assess the usefulness of “molecular grading fluorescence in situ hybridization (mG-FISH)” in a prognostic model for recurrence and progression using patients with non-muscle invasive bladder cancer.1 These were selected to be tumors of intermediate risk (according to the European Association of Urology definition), and FISH was carried out using multicolor probes to the centromeres of chromosomes 3, 7, 17 and the 9p21 locus. The authors found subclassification by mg-FISH into low risk (a diploid chromosomal pattern or only loss of 9p16 locus or chromosome 3 aneuploidy) and high risk (aneuploidy of chromosomes 7or 17) enabled a slight increase in the accuracy of the prognostic models, when compared with those derived using clinicopathological variables, such as tumor stage, grade, multiplicity and size, the patient's sex and age, and whether the patient received intravesical chemotherapy after resection. The study is notable for several reasons. Although it is retrospective in design, the authors used the UroVysion test as a prognostic tool (rather than diagnostic) to supplement the histopathological details of the tumor. As the authors suggested, patients with low mG-FISH could have less intensive and shorter treatment follow ups, which might reduce treatment costs. The downside to a less intensive surveillance strategy is potential delayed recognition of disease progression (which occurred in 16% of this cohort). This test causes no extra problems to a patient, as it is carried out in the urine sediment cells. Unfortunately, the relatively frequent problem (approximately 20%) is the insufficient number of cells (“no cells”) in the urine sediment specimen, which makes both the UroVysion test and the mG-FISH assessment impossible.2 As the proposed mG-FISH is treated as a supplement to the histopathological assessment of the tumor, perhaps it could be better to use this technique in the same tissue; that is, in the tumor, if, of course, after the initial recognition, this test is not carried out separately in order to monitor the disease. The authors underline that the use of the European Organization for the Research and Treatment of Cancer calculator, recommended by the European Association of Urology, did not allow selecting from the tested group those patients who will have disease recurrence or progression. It is a frequently mentioned imperfection of this calculator.3 Additionally, the introduction of the histopathological subclassifications might not be completely useful, and requires a great deal of experience in assessing specimens under the microscope. Bladder cancer is a heterogeneous disease – both interpatient and intratumor heterogeneity is important here.4 Hence, there are difficulties in finding markers that would be useful in routine practice. It is possible that heterogeneity itself and the variety of genetic changes are the reason for false negative results of the UroVysion test, whereas the histopathological recognition is positive. Therefore, undoubtedly, the proposed study shows an interesting approach, yet it requires analysis on a bigger and, what I find even more important, on a histopathologically homogeneous group of patients, so that clear conclusions can be drawn (patients with grade G3 should not be tested in the intermediate risk group). A similar approach with slightly different criteria for low- and high-risk positive FISH is presented in the work of Matsuyam et al.5 They found that the percentage of 9p21 loss (>12%) was an independent prognostic factor for recurrence, and the sum of the non-modal copy number fraction of chromosomes 3, 7 and 17 (>16%) was a prognostic factor for disease progression. None declared.

The reasons of false negative results of endobronchial ultrasound-guided transbronchial needle aspiration in the diagnosis of intrapulmonary and mediastinal malignancy.

Although the sensitivity can reach 99%, endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) has a significantly high false negative rate for diagnosis and staging of thoracic malignancy. We performed this retrospective study to investigate the causes of false negative results and to improve the efficacy and accuracy of EBUS-TBNA. We reviewed all patients suspected of intrapulmonary or mediastinal malignancy who undertook EBUS-TBNA between July 2009 and August 2012 in Chinese PLA general hospital. We retrieved the pathological results of EBUS-TBNA and video-assisted thoracic surgery (VATS) and follow-up data. The sensitivity, specificity, positive predictive value and negative predictive value were calculated. 185 patients were included in this study. Diagnosis of malignancy was established on 172 patients by EBUS-TBNA, and 8 patients with negative EBUS-TBNA result gained their final diagnosis of malignancy via VATS. The sensitivity, specificity, negative predictive value and accuracy for diagnosis of malignancy for EBUS-TBNA were 96%, 100%, 33% and 96% respectively. Inadequacy of the EBUS-TBNA specimens, internal necrosis in the lymph nodes and rare cancer types contributed to the false negative EBUS-TBNA results. VATS is obligatory to explain the negative results of EBUS-TBNA in patients suspected of malignancy.

Prospective, blinded comparison of cytology and DNA-image cytometry of brush biopsies for early detection of oral malignancy

Adjunctive techniques like DNA image cytometry (DNA-ICM) have been attributed to enhance the diagnostic performance of oral brush biopsies. The aim of the study was an evaluation of brush biopsies, analysed according to morphological criteria and by DNA-ICM vs. histological findings in a blinded prospective trial. Eighty eight brush biopsies of 70 patients were sampled. Only clinical suspicious but not evident malignant oral lesions were included. Clinical diagnosis was leukoplakia (n = 36), lichen planus (n = 18), verruciform erythroplakia (n = 12), erythroleukoplakia (n = 9), erosion (n = 7) and induration (n = 6). Evaluation was conducted via histology, cytology and DNA-ICM. Histological diagnosis revealed eight cases of squamous intraepithelial dysplasia (SIN 1 n = 6, SIN 2 n = 2), four cases of carcinoma-in situ and 25 cases of oral T1-cancer. Remaining cases were leukoplakia (n = 28), lichen planus (n = 15) and local inflammation (n = 8). Brush biopsy detected malignant lesions including SIN>1 with a sensitivity of 55% and a specificity of 100%. DNA-ICM had a sensitivity of 70% and a specificity of 100%. The combination of both methods showed a sensitivity of 76% and a specificity of 100%. The predominant reason for false negative results were sampling errors with insufficient cells (86% in brush biopsy and 100% in DNA-ICM). DNA-ICM has the potential to substantially improve the sensitivity of a pure morphological interpretation of oral brush biopsies. Method inherent sampling errors may be accountable for a lower sensitivity compared to conventional histological diagnosis. Therefore, DNA-ICM should not be used to rule out malignancy, when lesions are already clinically suspicious for oral cancer.

Comparative Evaluation of High-Throughput Small-Core (0.6-mm) and Large-Core (2-mm) Thyroid Tissue Microarray: Is Larger Better?

Tissue microarrays (TMAs) are useful in gene/protein expression profiling of large number of tumors. Several studies have validated that a 0.6-mm core of a large tumor would give results similar to results of the whole section. However, cores from colloid-filled thyroid follicles, for example in breast carcinoma, may contain fewer cells compared to solid tumors. The aim of this study is to validate thyroid TMAs choosing 2 core diameters, 0.6 and 2 mm, and to study the effect of core size and grid density on concordance with whole sections. 0.6-mm tissue cores were arrayed on a high-density TMA (406 cores). Two low-density TMAs (35 cores each) composed of 2-mm cores were also constructed. Immunohistochemistry was performed using primary antibodies to cytokeratin 19, HBME1, and CITED1 that have been found to be useful in the diagnosis of thyroid carcinoma. The results were compared with whole sections. The concordance between high-density TMAs and whole sections was 61 of 77 (79%) for cytokeratin 19; 76 of 80 (95%) for HBME 1; and 67 of 75 (89%) for CITED1. The concordance between the low-density TMAs and whole sections was cytokeratin 19, 41 of 51 (80%) for cytokeratin 19; HBME1, 52 of 56 (92.8%) for HBME1 and 58 of 59 (98%) for CITED1. The most frequent discordance was negative core but positive focal heterogeneous protein expression in whole sections. On whole sections, the sensitivity of tests increased but the specificity decreased compared to TMAs; however, the accuracy remained similar (77%-83%). Focal and heterogeneous protein expression was the most frequent reason for false negative results in TMAs. Tissue microarray remains an accurate method of screening for protein expression in a large number of thyroid tissues irrespective of core diameters or grid densities.

Intraoperative imprint cytology for the diagnosis of sentinel node metastases in breast cancer

Accurate intraoperative diagnosis of sentinel node metastases enables the selection of patients for axillary lymph node dissection, thus avoiding an unnecessary additional operation in patients with false-negative results. The main reason for false-negative results of imprint cytology was poor quality of the imprint samples because of sampling error. False-positive imprint cytology results that lead to an unnecessary axillary lymph node dissection are very rare. Imprint cytology can be recommended for the intraoperative examination of sentinel nodes in breast cancer patients.

MR arthrography in calcific tendinitis of the shoulder: diagnostic performance and pitfalls

The purpose was to assess the diagnostic performance of MR arthrography to diagnose calcific tendinitis of the shoulder and to assess the reasons for diagnostic errors. Standard MR arthrograms of 22 patients with calcific tendinitis and 61 controls were retrospectively analyzed by two independent and blinded radiologists. All cases were consecutively collected from a database. Conventional radiographs were available in all cases serving as gold standard. The supraspinatus was involved in 16, the infraspinatus in four and the subscapularis in two patients. All diagnostic errors were analyzed by two additional readers. Reader 1 correctly detected 12 of the 22 shoulders with and 42 of the 61 shoulders without calcific tendinitis (sensitivity 0.55, specificity 0.66). The corresponding values for reader 2 were 13 of 22 and 40 of 61 cases (sensitivity 0.59, specificity 0.69). Inter-rater agreement (kappa-value) was 0.42. Small size of the calcific deposits and isointensity compared to the surrounding tissue were the most important reasons for false negative results. Normal hypointense areas within the supraspinatus tendon substance and attachment were the main reason for false positive results. In conclusion, MR arthrography is insufficient in the diagnosis of calcific tendinitis. Normal hypointense parts of the rotator cuff may mimic calcific deposits and calcifications may not be detected when they are isointense compared to the rotator cuff. Therefore, MR imaging should not be interpreted without corresponding radiographs.

Tissue quality is an important determinant of telomerase activity as measured by TRAP assay.

Telomerase is a ribonucleoproteinwith the function of a DNA pol- ymerase, in which a segment of the RNAcomponent operates as an internal tem-plate. It adds hexameric (TTAGGG) re-peats to the telomeric ends of the chro-mosomes, thus compensating for thecontinued erosion of the telomeres thatoccurs in its absence (3,7). Reactivationof telomerase might be a necessaryevent for the sustained growth of mosthuman tumors (9). The development ofa polymerase chain reaction (PCR)-based telomerase assay, the telomericrepeat amplification protocol (TRAP),has permitted a large number of tumorsamples to be analyzed (5).Telomerase activity has been detect-ed in almost all human immortal celllines and in most human cancer tissues(2,5). However, some human tumor tis-sues seem not to express telomerase. Areview that compiled several studies ofhuman tumors indicated that tel- omerase activity occurred in human can-cer tissues in 75%–100% of cases, withan average of 84.8% (8). Furthermore,different studies on histologically simi-lar tumors revealed different propo- rtions of tumors with telomerase activ-ity. For example, in colorectal cancer,telomerase activity was detected in 32of 35 cases (92%) by Kim et al. (5), in40 of 50 cases (80%) by Li et al. (6)and in 8 of 8 cases (100%) by Yoshidaet al. (11). An explanation for these dis-crepancies could be heterogeneity ofhuman tumor tissues that invariablyconsist of mixtures of tumor cells andsurrounding stromal tissue. However,the TRAP assay has been shown to de-tect telomerase activity in as few as 1positive cell per 104cells (5). We pro-pose that some of the apparently neg-ative tumors yield RNA of insufficientquality and are therefore false negative. A second reason for false-negativeresults might be the presence ofTaq DNA polymerase inhibitors. To reco-gnize this type of problem, the TRAP as-say should be performed using an inter-nal control (10). The TRAP-ez eTelomerase Detection Kit (Oncor,Gaithersburg, MD, USA) includes theamplification of an internal control of36 bp in each assay, and a false-neg-ative result is concluded when the 36-bpamplified product is not observed. Thiscontrol, however, does not account forall apparently negative tumor tissues.Telomerase is a ribonucleoproteinenzyme that uses its RNA as a templatefor the synthesis of TTAGGG repeats atthe ends of the chromosomes (4). Forthis reason, telomerase activity necessi-tates an active protein and a nondegrad-ed RNA. These two conditions indicatethat the quality of the tissue is of vitalimportance for the success of thetelomerase detection assay. Strikingly,RNA quality control has never beenproposed to date. We therefore inclu-ded a control for RNA quality in ourTRAP assay protocol.We measured telomerase activity in50 colorectal cancer tissues derivedfrom 50 patients using the TRAP assay.About 10 mg of frozen tissue were h-omogenized in 150 µL of 1×CHAPS ly-sis buffer (5). The whole-tissue lysatewas rapidly frozen and stored at -80°C.The protein concentrations of the e- xtract were measured using the BC AProtein Assay Kit (Pierce Chemical,Rockford, IL, USA). Approximately0.1 µg of protein extracted from tissuewas used for each telomerase assay (25µL reaction). Forty-two samples (84%)were found to be positive in this assay,while 8 samples were telomerase-nega-tive. All 50 samples were taken fromthe tissue bank of the Institute of Pat-hology (Lausanne, Switzerland). Somesamples were not frozen immediately.This lapse of time that occurred som-etimes between surgery and freezing inliquid nitrogen is relatively important.Warm ischemia might account for tissueautolysis including RNA degradation,which can affect the telomerase activityresults. To test this hypothesis, totalRNA from these 50 colon cancer tissueswas extracted using TR

Detection of Human Papillomavirus (HPV) DNA in Cervical Swabs by the Polymerase Chain Reaction

The polymerase chain reaction (PCR) was used to detect human papillomavirus (HPV) type 16 DNA in cervical swabs from 37 patients with HPV 16-harboring cervical lesions (15 carcinomas and 22 cervical intraepithelial neoplasias). Primers amplifying a sequence of the human beta-globin genome were used for internal control together with the HPV 16-specific primers. The cell samples were prepared for PCR analysis by two different methods: either by phenol/chloroform extraction or by boiling in the presence of a chelating agent. HPV 16 DNA was found in 27 swabs. The detection rates were identical with both methods of preparation. Four of the 10 false-negative swabs contained too little DNA to permit amplification with the genomic primers. Excluding these insufficient samples, the detection rate was 82%. Reasons for false-negative results may include low cell numbers or failure to obtain cells representative of the underlying lesion. In conclusion, the PCR offers a satisfactory method of HPV detection in cervical swabs. Cell preparation can be restricted to simple boiling with a chelating agent. For optimal results, samples containing less than 2 x 10(4) cells should be discarded, and genomic primers should be used for internal control.

Solid-phase enzyme immunoassay for rotavirus antigen: Faecal protease activity as a reason for false-negative results

Rabbits and guinea pigs were immunized with purified bovine rotavirus. Immunoglobulin G fractions of the resulting antisera were used in a standard four-layer solid-phase enzyme immunoassay (EIA) for rotavirus antigen in human faecal specimens. Samples negative for rotavirus in electron microscopy, when diluted in standard EIA buffers, regularly gave absorbance values lower than those obtained with buffer blank only. By further diluting of the samples the resulting absorbance values were found to increase to the blank levels. When all dilution buffers were supplemented with 1–5% of bovine serum, negative samples at any dilution gave absorbance values close to those of the buffer blanks. Similar results were obtained if the serum was replaced by 1–5 mM of phenylmethylsulphonyl fluoride, a synthetic broad spectrum serine-type protease inhibitor. Aprotinin, another protease inhibitor, was without effect. A similar inhibition pattern was obtained when faecal specimens were tested in a caseinolytic quantitative protease assay in the presence of the above inhibitors. These observations suggest that protease activity present in human faecal samples may cause false-negative results in solid-phase immunoassay for viral antigens, unless appropriate means are used to avoid this interference.

Technical problems in arthrography of the painful hip arthroplasty

Arthrography following hip replacement has been reviewed. The problems encountered have been analysed with particular reference to technique and accuracy. The accepted criteria for loosening have not been consistently reliable and have led to false positive interpretation of loosening of the acetabular component. The reasons for false negative results are also considered.