Nooreldin Zendehrokh

Preparation of DNA from cytological material: Effects of Fixation, Staining, and Mounting Medium on DNA Yield and Quality.

Personalized oncology requires molecular analysis of tumor cells. Several studies have demonstrated that cytological material is suitable for DNA analysis, but to the authors knowledge there are no systematic studies comparing how the yield and quality of extracted DNA is affected by the various techniques used for the preparation of cytological material.

Telomere repeat amplification protocol (TRAP) in situ reveals telomerase activity in three cell types in effusions : malignant cells, proliferative mesothelial cells, and lymphocytes

Telomerase Repeat Amplification Protocol (TRAP) in situ was performed on cytospin preparations from 65 effusions from the serous cavities (45 pleural and 19 ascitic fluids and one pericardial fluid) submitted for routine diagnosis and the results were correlated to cytological morphology. Three types of cells with nuclear fluorescence were identified: malignant cells, hyperplastic mesothelial cell and lymphocytes. Of 38 cytologically malignant effusions, 12 showed strong reactivity in all malignant cells, three strong reactivity in part of the malignant population, whereas 12 showed moderate reactivity in the whole and five in part of the malignant population, respectively. In five malignant effusions weak reactivity was found in all (one case) and in scattered (four cases) malignant cells. Two effusions contained telomerase-negative malignant cells. Two pleural and two ascitic fluids contained proliferative mesothelial cells with weak or, in one case, moderate reactivity. Lymphocytes usually showed weak telomerase activity. Telomerase was expressed in almost all malignant tumours metastatic to serous cavities. Heterogeneity in tumour populations was demonstrated, which may have diagnostic implications, especially in cytology. Weak or moderate reactivity was found in lymphocytes and in some mesothelial proliferations and may explain the low specificity for malignancy sometimes obtained with the TRAP extract method. The weak reactivity found in lymphocytes may reduce the specificity when the extract method is used but causes no diagnostic problem with the TRAP in situ method.

Liquid‐based cytology using cytorich red/tripath is diagnostically equivalent to conventional smears for bronchial washings and brushings and reduces the cost

A split sample study was performed on 109 bronchial brushings and washings and evaluated from conventional slides (CS) and CytoRich Red/Tripath preparations (CRR/Tripath). Unassessable bronchial washings were significantly more frequent in CS (5 vs. 0), but as all brushings were assessable with both methods, no overall diagnostic advantage was found. CS and CRR/Tripath gave discordant diagnoses in one case with a final benign diagnosis and in six cases with final malignant diagnoses. In the benign case, atypia was assessed in CS. In the malignant cases, suspected malignancy was found in one CRR/Tripath and one CS, atypia vs. benign assessment was also balanced, with three atypias in CRR/Tripath and two in CS. The better preserved cells in CRR/Tripath facilitated correct diagnosis in some cases, but might also lead to false positive diagnoses. In small cell carcinomas diagnostic hints such as smearing and moulding were less pronounced in CRR/Tripath but this did not affect the diagnostic accuracy. Overall, the diagnostic performance with CRR/Tripath was at least as good as with conventional slides, although statistically no difference could be seen. The number of slides and screening time, and thereby cost was significantly reduced with CRR/Tripath, thus the liquid‐based method is preferred for bronchial washings and brushings. Diagn. Cytopathol. 2013;41:876–884.© 2013 Wiley Periodicals, Inc.

Comparison of Liquid-Based Cytology and Conventional Filter Preparations in Urine: A Possible Serious Pitfall

To the Editors: We read with interest the report by Sng et al1 on the comparison of Cytospin and ThinPrep processing of urinary cytology, published in Acta Cytologica. We have compared ThinPrep to the membrane filter preparation method currently in use in our laboratory for urinary cytology, and we have come to conclusions similar to those of Sng et al.1 ThinPrep was applied to 47 urinary specimens (22 voided urine, 10 bladder washings and 15 bladder urine) submitted fresh to the Department of Clinical Cytology and Pathology, Malmö University Hospital, and its diagnostic performance and the effect on diagnostic criteria were compared to membrane (PCTE MEMB) filter (Millipore/NovAseptic AB, SE-449 34, Nödinge, Sweden) preparations. Each specimen was divided into 2 aliquots, and 1 aliquot was used to make 1 membrane filter preparation slide. Briefly, the filter is soaked in 95% ethanol, applied to the top of the slides and fixed in a special holder and the fresh specimen poured onto the filter without prior centrifugation. The other aliquot was used to process ThinPrep slides according to the instruction of the manufacturer (Cytyc, Marlborough, Massachusetts, U.S.A.).

Comparison of NCL-hTERT Antibody Reactivity and Telomere Repeat Amplification Protocol in Situ in Effusions

OBJECTIVEnTo compare the performances of 2 methods, telomerase repeat amplification protocol (TRAP) in situ and antibodies to the hTERT protein, in assessing telomerase activity.nnnSTUDY DESIGNnTRAP in situ and immunohistochemistry with a commercial antibody (NCL-hTERT) was performed on 54 body cavity effusions. The results were compared and correlated to diagnosis.nnnRESULTSnThirty-four effusions from patients with verified malignant disease contained cytologically malignant cells. Both methods were positive in 33 of the cases, whereas only hTERT was positive in 1 case. Twenty effusions, all containing mesothelial cells, came from patients with benign conditions. In 2 fluids atypical, hyperplastic mesothelial cells were both TRAP in situ and hTERT positive. All remaining 18 fluids were TRAP in situ negative, whereas 12 of 18 were hTERT positive. Thus the results of TRAP in situ and hTERT immunohistochemistry disagreed in 1 of 34 (3%) malignant and 12 of 20 (60%) benign cases.nnnCONCLUSIONnThe sensitivities for malignancy were similar for TRAP in situ and hTERT immunohistochemistry. The specificity of the applied hTERT antibody was significantly lower, due to hTERT reactivity in mesothelial cells.

Weak telomerase activity in malignant cells in metastatic serous effusions - Correlation to short survival time

OBJECTIVEnThe purpose of this study was to evaluate whether the intensity of telomerase activity measured on the cellular level in effusions correlates to survival time in cases of metastatic spread to the serous cavities.nnnSTUDY DESIGNnTelomere repeat amplification protocol (TRAP) in situ was applied to 46 effusions containing metastatic cancer cells.nnnRESULTSnWeak telomerase activity in tumor cells was seen in 7 cases, and moderate or strong activity in 39 cases. The intensity of the enzyme activity correlated significantly to survival (Kaplan-Meier survival analysis), the median survival time being 18 days for patients with weakly positive tumors and 100 days for patients with moderately or strongly positive tumors (Kruskal-Wallis test, p = 0.021).nnnCONCLUSIONnThe strong relationship between telomerase activity in tumor cells in effusions and survival time has never been described before. Determination of telomerase activity on the cellular level provides a way to identify a subgroup of patients with a high probability of short survival.