Agreement and internal quality assurance of the Neubauer hemocytometer and Makler chamber for human sperm concentration determination.

OBJECTIVE: The Neubauer hemocytometer, as well as the Makler chamber, are devices commonly used in andrology laboratories. The present study aimed to verify if both methods yield comparable results, and whether they can be used interchangeably to determine sperm concentration. METHODS: Sperm and latex beads concentration measurements were performed with the Neubauer hemocytometer and the Makler chamber. Fixed and proportional biases were estimated, and the method agreement was determined by assessing sperm concentration results with the Bland and Altman plot. The Coefficient of Variation (CV) and relative bias were calculated as an index of precision and accuracy, respectively, by measuring latex beads target concentrations in both chambers. RESULTS: The Makler chamber systematically overestimated the Neubauer hemocytometer concentration measurements by a mean of -7.99%, with limits of agreement (LOA) between -41% to 25.61% (p<0.001). The fixed bias was found for concentration values inferior to 40 x 106/ml range (p<0.001), but not higher concentration results (p>0.05). Measurements with the Neubauer hemocytometer showed the greatest consistency in the study with the CV ranging from 3.01% to 6.67%; while the CV with the Makler chamber ranged from 8.46% to 25.64%. The relative bias for the Neubauer hemocytometer determinations varied from 0.12% to 8.40%, while for the Makler chamber varied from 7.6% to an overestimation of 38.0%. CONCLUSIONS: Measurements made with the Makler chamber demonstrated more variability and a higher degree of overestimation. The Makler chamber is a poor substitute to the Neubauer hemocytometer for evaluation of oligozoospermic samples, although both chambers render similar results for highly concentrated samples.

Role of DNA ploidy in diagnosis and prognosis of high-grade cervical intraepithelial neoplasia: A prospective cohort study.

OBJECTIVE: To compare the sensitivity and specificity of DNA ploidy with cytology, human papillomavirus (HPV) testing and colposcopy in diagnosis of high-grade cervical intraepithelial neoplasia (CIN) and to assess the role of aneuploidy in cervical lesions with the worst prognosis. A prospective observational cohort study was conducted on 254 women with altered colpocytology. METHODS: Colposcopy, biopsy, DNA-ICM and HPV examinations were applied to cervical cytological and histological samples. Participants were evaluated every 6 months and divided into two groups: 'Harm' and 'No-harm'. Logistic regression and multivariate COX model were used to identify independent risk factors for diagnosis and prognosis of high-grade CIN, and ROC curve to assess the sensitivity and specificity of methods. RESULTS: Variables 'age greater than or equal to 30 years', 'lesion size greater than 20%', 'aneuploidy' and 'HPV 16' were associated with diagnosis of high-grade CIN and 'aneuploidy' and 'women living with HIV', with a worse prognosis. Agreement for colposcopy was good, with a sensitivity of 79.3% and specificity of 94.4%; DNA-ICM and cytology were moderate, with sensitivity of 74.6% and 72.3% and specificity of 85.3% and 76.1%, respectively. High-risk HPV and HPV 16 tests were weak, with sensitivity of 75.0% and 43.75% and specificity of 50.0% and 88.64%, respectively. CONCLUSIONS: In relation to high-grade CIN diagnosis, DNA-ICM presented similar sensitivity and specificity to cytology and high-risk HPV test when associated with HPV 16. Regarding prognosis, this research certifies that aneuploidy is considered a predictor of more severe cervical injury.

DNA-ICM as an adjuvant method applied on oral cytological specimens.

OBJECTIVE: This study aimed to evaluate cytology diagnosis accuracy using adjuvant methods in clinical routine for oral cancer. STUDY DESIGN: This prospective study was conducted on 98 patients with clinically potentially malignant or malignant oral cavity lesions. One oral lesion smear was taken from each patient using a cytobrush before biopsy and stored at PreservCyt Thinprep. Samples were cytologically analyzed, and DNA ploidy measurement was performed on the same slide. The diagnostic methods' accuracy was then calculated. RESULTS: In clinical inspection, 61 patients had suspicious lesions for malignancy, whereas 37 had potentially malignant disorders. Cytology associated with DNA image cytometry presented a sensitivity of 81.2% and specificity of 90.9%. When analyzing lesions located in high-risk sites to oral malignancies individually, cytology associated with DNA image cytometry presented a sensitivity of 88.2%, specificity of 100.0%, accuracy of 90.0%, and Kappa value of 0.77 (CI 95%: 0.48-1.00). CONCLUSIONS: Association between cytology and DNA image cytometry is an objective and non-invasive diagnostic method that demonstrated high sensitivity and specificity in diagnosing malignant epithelial squamous cell transformation in the oral cavity.

DNA image cytometry parameters to identify high-grade cervical lesions.

OBJECTIVE: Evaluate the performance of different DNA image cytometry (DNA-ICM) ploidy parameters in the categorisation of DNA-ICM results and identification of high-grade cervical intraepithelial neoplasia or worse (≥ CIN2). METHODS: Cervical samples from 232 women were collected for DNA-ICM analysis and biopsy confirmation. Five DNA parameters were used to define DNA aneuploidy: number of cells with exceeding events (EE) over 2.5cEE, 4cEE, 5cEE and 9cEE, and aneuploid stemlines. DNA-ICM results were categorised as normal, suspicious, and abnormal. RESULTS: For individual DNA ploidy parameters, sensitivity values for 50 cells with 2.5cEE, 45 cells with 4cEE, 1 cell with 9cEE and aneuploid stemline were 72.95%. 54.1%, 69.67% and 54.1%, while specificity values were 80.0%, 90.0%, 89.09% and 95.45%, respectively. For the 5cEE parameter, the sensitivity values for 1, 2, 3, 4 and 5 cells were 93.44%, 85.25%, 81.97%, 77.87% and 75.41%, while specificity values were 46.36%, 63.64%, 74.55%, 76.36% and 80.91%, respectively. For categorised DNA-ICM results, a suspicious result showed superior sensitivity than an abnormal result (87.70% vs 82.79%, P = 0.031), but lower specificity (54.55% vs 75.45%, P < 0.001). Both types of DNA-ICM result were statistically significantly different from a normal result (P < 0.05). CONCLUSION: For prognostic purposes, 1 cell with 9cEE, 45 cells with 4cEE and aneuploid stemline are the best parameters with which to categorise an abnormal DNA-ICM result, followed by 50 cells with 2.5cEE and 4 cells with 5cEE. For screening purposes, 10 cells with 2.5cEE, 10 cells with 4cEE, and 2 cells with 5cEE are suitable parameters with which to categorise a suspicious DNA-ICM result.

Aneuploidy detection for diagnostic and prognostic use in premalignant and malignant lesions of the uterine cervix: A systematic review.

OBJECTIVE: To systematically review the role of aneuploidy detection alone or in combination with other methods in cervical cancer screening and to evaluate the value of aneuploidy to predict the behavior of premalignant cervical lesions. METHOD: We conducted a systematic review based on an electronic search for articles published between 2001 and 2020 across databases including MEDLINE/PubMed, Scopus, and Web of Science. Studies were subjected to data extraction, risk of bias, and narrative synthesis. RESULTS: A total of 15 articles were included in the review. Eight out of 15 studies (53.3%) were judged to be at a high or unclear risk of bias. From the 15 included studies, the index test to detect aneuploidy was DNA image cytometry (DNA-ICM) in 12 studies and DNA flow cytometry (DNA-FCM) in three studies. Nine studies also evaluated the performance of cytology and/or human papillomavirus (HPV) tests. For DNA-ICM, sensitivity to detect cervical intraepithelial neoplasia or worse (CIN2+) varied between 59.0% and 95.9% and specificity varied between 54.1% and 100%. For DNA-FCM, sensitivity varied between 27.3% to 96.8% and specificity was 100%. For cytological evaluation, sensitivity varied between 25.0% and 70.4% and specificity varied between 70.6% and 99.9%. For HPV detection, sensitivity varied between 39.4% and 100% and specificity varied between 23.3% and 84.3%. CONCLUSION: DNA ploidy along with atypical cells findings in cytology and/or HPV detection revealed great value to detect CIN2+ lesions and to predict which lesions are more likely to progress to cervical cancer.

DNA ploidy measurement and human papillomavirus in abnormal cervical cytology.

OBJECTIVE: To compare the efficacy of high-risk human papillomavirus (HR-HPV) and DNA image cytometry (DNA-ICM) status for identifying high-grade cervical intraepithelial neoplasia or worse (≥CIN2). METHODS: This cross-sectional study was performed in women undergoing follow-up procedure after a previous abnormal cervical cytology. Cervical cells were collected for HPV detection and DNA ploidy measurement. Biopsy samples were taken for histological confirmation. Sensitivity and specificity values for ≥CIN2 detection with HR-HPV and DNA-ICM were determined. RESULTS: HR-HPV was present in 74.5% of the women. The most frequent HPV infection was HPV 16, followed by HPV 31, 33 and 58. Aneuploidy was observed in 60.6% of all cases. Referral cytology revealed 78.0% sensitivity and 68.6% specificity for detecting a ≥CIN2 lesion. The HR-HPV test alone showed 92.7% sensitivity, albeit it was not statistically different from DNA-ICM (88.1%, P > .05). Positivity for HPV or DNA-ICM resulted in 100% sensitivity. Higher specificity was observed for the combination of HR-HPV and DNA-ICM (88.6%), with no difference from DNA-ICM alone (85.7%, P > .05). CONCLUSION: DNA-ICM or HR-HPV positivity identified all cases of ≥CIN2 in women undergoing follow-up procedure after a previous abnormal cervical cytology. Routine cervical cancer screening could be improved by the incorporation of DNA-ICM as a complementary method to primary screening to identify which women need closer follow-up.

Comparative evaluation of the GP5+/6+, MY09/11 and PGMY09/11 primer sets for HPV detection by PCR in oral squamous cell carcinomas.

The aim of this study was to evaluate the use of GP5+/6+, MY09/11 and PGMY09/11 primer sets for the detection of human papillomavirus (HPV) DNA by single step polymerase chain reaction (PCR) and nested PCR in formalin-fixed and paraffin-embedded (FFPE) tissues from oral squamous cell carcinomas (OSCCs). DNA extracted from FFPE tissues were tested for amplification of the human beta globin gene with PCO3/4 primers. Positive samples for this gene were tested for HPV DNA using single step PCR with GP5+/6+, MY09/11 and PGMY09/11 primer sets. All negative samples at single step PCR with MY09/11 and PGMY09/11 were subjected to a further PCR with GP5+/6+ primers using the non-amplified product in the previously reactions (nested PCR) as samples. Among 26 samples, 23 were positive for the human beta globin gene and were considered viable for HPV DNA detection by PCR. Single step PCR with GP5+/6+ and MY09/11 primers and MY/GP+ nested PCR did not amplify HPV DNA in any samples. PGMY09/11 primers detected HPV DNA in 13.0% of OSCC cases and this rate was raise to 17.4% with the use of PGMY/GP+ nested PCR. According to our results the PGMY/GP+ nested PCR is the most appropriate primer set for the detection of HPV DNA using FFPE samples from OSCC.

Detecção de HPV utilizando iniciadores MY09/MY11 e GP5+/GP6+ em pacientes com alterações citológicas e/ou colposcópicas / HPV detection using primers MY09/MY11 and GP5+/GP6+ in patients with cytologic and/or colposcopic changes

Introduction: Cervical cancer is one of the most common diseases among women, and cause considerable morbidity and mortality. Considering that cervical cancer is an important neoplasia in northeastern Brazil, and the prevalence of high-risk human papillomavirus (HPV) is directly associated with it, this work had aimed to correlate the cytological and/or colposcopic findings with HPV infection status, and verify the performance of MY09/MY11 and GP5+/6+ primers for HPV detection. Material and method: Patients in this study were from Penedo-AL, a city with high level of poverty (poverty rate of 60.62%). Out of 70 patients with cytological and/or colposcopic changes, 32 agreed to participate in the study. Results: Regarding cytology, 21 (30%) patients presented atypical squamous cells of undetermined significance (ASC-US); 20 (29%), atypical glandular cells of undetermined significance (AGUS); 12 (17%), low-grade intraepithelial lesion (LSIL); five (7%), high-grade intraepithelial lesion (HSIL); and 12 (17%), positive colposcopy. From these, 27 (84%) presented the band gene encoding for human ß-globin. From the 27 patients, eight (30%) were positive for HPV. The results showed that the deoxyribonucleic acid (DNA) of HPV was detected in 15% and 30% by using MY-PCR and GP +-PCR, respectively. Conclusion: This study suggests that more than one type of oligonucleotide primer should be used in clinical samples to increase sensitivity for the detection of HPV...

Introdução: O câncer cervical é uma das doenças mais frequentes entre mulheres, e causa considerável morbidade e mortalidade. Com base nos fatos de que o câncer cervical é uma neoplasia importante no nordeste brasileiro, e que a prevalência do papilomavírus humano (HPV) de alto risco está diretamente associado a ele, este trabalho teve como objetivos correlacionar os achados citológicos e/ou colposcópicos com status de infecção de HPV e verificar o desempenho dos iniciadores MY09/MY11 e GP5+/GP6+ para detecção do HPV. Material e métodos: Os pacientes deste estudo foram de Penedo-AL, uma cidade com elevado nível de pobreza (índice de pobreza de 60,62%). Do total de 70 pacientes com alterações citológicas e/ou colposcópicas, 32 aceitaram participar do estudo. Resultados: Com relação a citologia, 21 (30%) pacientes apresentaram células escamosas atípicas de significado indeterminado (ASC-US); 20 (29%), células glandulares atípicas de significado indeterminado (AGUS); 12 (17%), lesão intraepitelial de baixo grau (LSIL); cinco (7%), lesão intraepitelial de alto grau (HSIL); e 12 (17%), colposcopia positiva. Destas, 27 (84%) apresentaram banda do gene que codifica para ß-globina humana. Das 27 pacientes, oito (30%) apresentaram positividade para o HPV. Os resultados mostraram que o ácido desoxirribonucleico (DNA) do HPV foi detectado em 15% e 30%, usando MY-PCR e GP +-PCR, respectivamente. Conclusão: Este estudo sugere que mais de um tipo de iniciador de oligonucleotídeo deve ser utilizado em amostras clínicas para aumentar a sensibilidade na detecção do HPV...

Evaluation of adherence to treatment by patients seen in a psychosocial care center in northeastern Brazil

In the treatment of mental disorders, nonadherence to medication, the main cause of psychiatric morbidity, is observed in about 50 percent of the cases and is responsible for numerous losses. This study evaluated adherence to drug treatment by patients seen in a Psychosocial Care Center (CAPS) in northeastern Brazil. Adherence to treatment was evaluated using the Haynes-Sackett and Morisky-Green-Levine tests. All patients registered in the CAPS were included in the study (n= 101). Only 11.88 percent of the patients adhered to drug treatment. The main reasons not to use medication were: oblivion (68.83 percent), feeling unwell after taking the medication (54.22 percent), not having money to buy the medication (43.83 percent), not finding the medication in the public health service (39.94 percent) and fear of harm that might be caused by the drug (28.90 percent). Furthermore, 85.1 percent of the patients did not know their diseases, 88.1 percent did not know their treatment, 86.4 percent did not feel good when they took their medication, and 88.1 percent took their medication incorrectly. The results revealed that the lack of information about diseases and drugs used, the nuisance posed by drug therapy and the low access to medications reduce adherence to treatment and, consequently, treatment effectiveness.

No tratamento de desordens mentais, a não-adesão ao tratamento ocorre em cerca de 50 por cento dos casos e é responsável por inúmeros prejuízos, além de ser a principal causa de morbidade psiquiátrica. O presente estudo objetivou avaliar a adesão ao tratamento medicamentoso de pacientes atendidos em um Centro de Atenção Psicossocial (CAPS) no Nordeste do Brasil. A avaliação da adesão ao tratamento foi feita através dos testes de Haynes-Sackett e Morisky-Green-Levine. Todos os pacientes cadastrados no CAPS foram incluídos no estudo (n = 101). Observou-se que apenas 11,88 por cento dos pacientes aderiram ao tratamento medicamentoso. As principais razões para o não uso dos medicamentos foram: esquecimento (68,83 por cento), sentir-se mal após a ingestão de medicamentos (54,22 por cento), não ter dinheiro para comprar medicamentos (43,83 por cento), não encontrar os medicamentos no serviço público de saúde (39,94 por cento) e medo dos danos causados pelos medicamentos (28,90 por cento). Além disso, observou-se que 85,1 por cento dos pacientes não conheciam suas doenças, 88,1 por cento não conheciam seus tratamentos, 86,4 por cento não se sentiam bem quando usavam medicamentos e 88,1 por cento usavam os medicamentos incorretamente. Os resultados demonstram que a falta de informação sobre doenças e medicamentos, os danos decorrentes da terapia medicamentosa e o baixo acesso aos medicamentos comprometem a adesão ao tratamento e, consequentemente, a eficácia do tratamento.

9p21 Deletion in the diagnosis of malignant mesothelioma in serous effusions additional to immunocytochemistry, DNA-ICM, and AgNOR analysis.

BACKGROUND: The diagnosis of malignant mesothelioma (MM) in serous effusions is difficult but may be achieved by the application of adjuvant methods. METHODS: The authors cytologically diagnosed 33 effusions as suspicious or positive for MM cells by using DNA-image cytometry (DNA-ICM), immunocytochemistry and AgNOR analysis. The authors further detected 9p21 deletions by chromosomal fluorescence in situ hybridization (FISH). In addition, 31 cases of metastatic carcinomas and 39 of tumor cell-negative effusions were investigated. All diagnoses were confirmed by histologic and/or clinical follow-up. RESULTS: DNA aneuploidy was found in 71% of MMs, 100% of metastatic carcinomas, and in none of the negative effusions. Calretinin was positive in 100% of MMs, in none of the metastatic carcinomas, and in 94.9% of negative effusions. BerEP4 showed positivity in 15.6% of MMs, 87.1% of metastatic carcinomas, and in none of the negative effusions. With AgNOR analysis, 89.3% of MMs and 96.7% of metastatic carcinomas showed >or=2.5 AgNOR dots as satellites and >or=4.5 as total AgNOR counts. 9p21 deletions were demonstrated in 90.9% of MM cases, 45.2% of metastatic carcinomas, and in none of the negative effusions. By cytology alone, 81.8% of MMs were identified unequivocally. Addition of DNA-ICM improved the prevalence of tumor cell detection to 87.9% and of AgNOR analysis to 97%. The introduction of 9p21 deletions by FISH improved this prevalence to 100%. CONCLUSIONS: Because of these results, the authors propose the sequential application of immunocytochemistry, DNA-ICM, and AgNOR analysis to establish a cytological diagnosis of malignant mesothelioma in serous effusions. In persistent doubtful diagnoses, the authors recommend fluorescence in situ hybridization to analyze the 9p21 deletion.

Immunocytochemical diagnosis of hepatocellular carcinoma and identification of carcinomas of unknown primary metastatic to the liver on fine-needle aspiration cytologies.

BACKGROUND: Difficulties with cytologic diagnoses on fine-needle aspiration cytology (FNAC) of the liver can be overcome by the application of immunocytochemical panels applied on smears. The aim of the current study was to analyze the performance of a panel of monoclonal antibodies to differentiate hepatocellular carcinoma (HCC) from metastatic carcinoma (MC) or regenerative nodules, and to identify the to date unknown primary sites of carcinomas that had metastasized to the liver. METHODS: In a validating cohort study, 108 FNACs coin lesions in the liver were routinely evaluated applying immunocytochemistry as an ancillary method. All patients had confirmatory histologic and/or clinical follow-up. A total of 23 HCCs were analyzed for the distinction from MC or regenerative nodules applying a panel of HepPar1, alpha-fetoprotein, BerEP4, CD31, CD68, and Ki-67. A total of 85 cases of unknown primary tumor metastatic to the liver were used to identify the tumor sites applying a panel of CK5/6, CK7, CK20, CA 125, thyroid transcription factor-1 (TTF-1), and Cdx2. RESULTS: Typing accuracy to differentiate HCC from MC or regenerative nodules was 100% and 90.3%, respectively, to identify the primary tumor site of MC. In 23 cases, the site of the primary tumor remained clinically unknown. CONCLUSIONS: The application of immunocytochemical panels on the same slide used for microscopic diagnosis is a useful tool in the routine assessment of FNACs of the liver to discriminate HCCs from MC or regenerative nodules and for the identification of primary sites of MC. Their performance should be confirmed in a larger series of cases.

Methylation assay for the diagnosis of lung cancer on bronchial aspirates: a cohort study.

PURPOSE: Recent studies have detected aberrant promoter methylation of adenomatous polyposis coli promoter 1 A (APC), cyclin-dependent kinase inhibitor-2A (p16(INK4a)), retinoic acid receptor beta2, and RAS association domain family protein 1 (RASSF1A) in bronchial aspirates and suggested their use as biomarkers for lung cancer diagnostics. The purpose of this study was to validate these candidate marker genes in a retrospective cohort study. EXPERIMENTAL DESIGN: Bronchial aspirates collected from a cohort comprising 247 patients with suspected lung cancer were investigated retrospectively regarding aberrant promoter methylation using a quantitative methylation-specific real-time PCR (QMSP). RESULTS: Eighty-nine patients were diagnosed with primary lung cancer, 102 had benign lung disease, and 56 showed miscellaneous other conditions. A panel consisting of APC, p16(INK4a), and RASSF1A emerged as useful combination. This panel detected aberrant methylation in bronchial aspirates of 22 of 35 (63%) and 21 of 44 (44%) centrally and peripherally located primary lung cancers, respectively. Bronchial aspirates also showed aberrant methylation in 5 of 7 (71%) patients with a recurrent lung cancer and in 8 of 30 (27%) cases without tumor recurrence. In contrast, only 1 of 102 patients with benign lung disease displayed a (false) positive test result. Rarely, aberrant methylation was found in patients with other malignancies (3 of 16). The QMSP assay correctly confirmed lung cancer in 8 of 12 (67%) cases with an ambiguous cytology. Moreover, it disclosed 9 of 26 (35%) of peripheral tumors lacking simultaneous cytologic or histologic diagnosis of malignancy. CONCLUSIONS: Our findings suggest that the QMSP assay could be applied as a reflex test in cases of suspected lung cancer that defy a definite diagnosis by conventional methods. Thus, the assay could be a useful diagnostic adjunct especially regarding peripheral tumors.

Utilidade da análise de DNA por citometria de fluxo em fluidos da cavidade corporal em comparaçäo com a citologia convencional / Utility of DNA flow cytometric analysis of body cavity fluids in comparison with conventional cytology

A potencial utilidade da análise por citometria de fluxo em rotina de citologia diagnóstica para efusöes foi avaliada com 104 amostras de fluidos da cavidade corporal. A citologia convencional mostrou näo positividade (0/33) para efusöes de pacientes com doenças näo malignas e 45 porcento de positividade (38/71) para efusöes de pacientes com doenças malignas. Inversamente, a citometria de fluxo mostrou näo positividade (0/33) de pacientes com doenças näo malignas e 18 porcento de positividade (padräo aneuplóide) (13/71) com doenças malignas. Em 13 casos examinados, 8 casos (62 porcento) mostraram idêntico padräo de ploidia de DNA entre o local da efusäo e o tumor primário. A citometria de fluxo mostrou positividade em 4 amostras citologicamente negativas (3 linfomas malignos e 1 carcinoma de células claras). Nos casos com linfomas malignos, a citologia convencional mostrou 40 porcento de positividade (4/10) e a citometria de fluxo 60 porcento (6/10) de positividade. Estes resultados indicam que a análise de DNA de células em efusöes malignas näo tem valor diagnóstico como um teste de rotina, mas pode ter nos casos de linfoma maligno