Detection Of HTERT MRNA Of Cells Collected From Pleural Fluid By The In Situ Hybridization Method: Clinical Value For The Differential Diagnosis Between Benign And Malignant Pleural Effusions

OBJECTIVE: Using the conventional cytologic examination of pleural fluid as contrast, this research was to evaluate the clinical value of detection of hTERT mRNA of cells collected from pleural fluid by the in situ hybridization (ISH) method for the differential diagnosis between benign and malignant pleural effusions. METHODS: Firstly, the experimental method for detection of hTERT mRNA of cells collected from pleural fluid by a two-stage ISH approach using digoxin-labelled oligonucleotide probes was established. The main process was that we performed the procedure of hybridization in situ between hTERT mRNA of cells and digoxin-labelled oligonucleotide probes after having made cells smears and fulfilled pretreatment. The hTERT mRNA signals could be detected by the following antigen-antibody-enzyme-dye pathway. With negative and positive contrast and manipulation in hypothermic state, the experimental study was independent of clinical diagnosis and treatment. Secondly, the prospective study for detection of hTERT mRNA of cells collected from pleural fluid of 103 random cases with pleural effusions diagnosed initially has been completed. For each of the 103 cases, pleural fluid was reserved for this laboratory research and clinical cytologic examinaion simultaneously when the first thoracentesis was performed. For every case whose result of the first cytologic examination was negative, if the possibility of malignancy couldn't be ruled out, clinicians would take cytologic examination of pleural fluid repeatedly (at least 3 times) and other examination in order to obtain a definite etiological diagnosis. After having accomplished all the experiments, we compared the experimental results of detection of hTERT mRNA of cells collected from pleural fluid by ISH with the relevant clinical data such as the results of cytologicexamination and the outcome of follow-up. RESULTS: The experimental method for detection of hTERT mRNA of cells collected from pleural fluid by a two-stage ISH approach using digoxin-labelled oligonucleotide probes had been established successfully. The experimental results were consistent with the theoretical expectations on the whole. In most of the cases with malignant pleural effusions, we could find the cells with cytoplasm dyed deep and a little nucleus colored under microscope irrespective of cytomorphological findings. On the contrary, in most of the cases with benign pleural effusions, there wasn't any cell with pigmentation under microscope. So, the criteria to judge positive and negative results of experiment were established. Out of the 103 cases, according to the final diagnosis, there were 41 cases with malignant pleural effusions, 55 cases with benign pleural effusions and 7 cases with pleural effusions of equivocal origins. When the 7 cases of indefinite etiology were excluded in the analysis of results, there were significantly statistical difference between the positive rate of detection of hTERT mRNA of cells collected from pleural fluid by ISH in malignant group and that in benign group (P<0.001). By this method, the diagnostic sensitivity (SN) for malignant pleural effusions was 80.49%, the specificity (SP) was 94.55%, the accuracy (AC) was 88.54%, the positive predictive value (PPV) was 91.67%, the negative predictive value (NPV) was 86.67%. Clinically, the indices of diagnostic efficacy for malignant pleural effusions of the first cytologic examination of pleural fluid were: SN 29.27%, SP 100%, AC 69.79%, PPV 100%, NPV 65.48%; the indices of diagnostic efficacy of the repeated cytologic examinations of pleural fluid were: SN 51.22%, SP 100%, AC 79.17%, PPV 100%, NPV 73.33%. If the laboratory detection of hTERT mRNA of cells collected from pleural fluid by ISH and the clinically repeated cytologic examinations of pleural fluid were combined, and if we determine a case with pleural effusion as malignant one when any result of the two methods was positive, whereas benign pleural effusion was judged when both results of the two methods were negative, the indices of diagnostic e...