Objective: To evaluate the clinical value of the MeltPro MTB assays in the diagnosis of drug-resistant tuberculosis. Methods: A cross-sectional study design was used to retrospectively collect all 4 551 patients with confirmed tuberculosis between January 2018 and December 2019 at Beijing Chest Hospital, Capital Medical University. Phenotypic drug sensitivity test and GeneXpert MTB/RIF (hereafter referred to as "Xpert") assay were used as gold standards to analyze the accuracy of the probe melting curve method. The clinical value of this technique was also evaluated as a complementary method to conventional assays of drug resistance to increase the detective rate of drug-resistant tuberculosis. Results: By taking the phenotypic drug susceptibility test as the gold standard, the sensitivity of the MeltPro MTB assays to detect resistance to rifampicin, isoniazid, ethambutol and fluoroquinolone was 14/15, 95.7%(22/23), 2/4 and 8/9,respectively; and the specificity was 92.0%(115/125), 93.2%(109/117), 90.4%(123/136) and 93.9%(123/131),respectively; the overall concordance rate was 92.1%(95%CI:89.6%-94.1%),and the Kappa value of the consistency test was 0.63(95%CI:0.55-0.72).By taking the Xpert test results as the reference, the sensitivity of this technology to the detection of rifampicin resistance was 93.6%(44/47), the specificity was100%(310/310), the concordance rate was 99.2%(95%CI:97.6%-99.7%), and the Kappa value of the consistency test was 0.96(95%CI:0.93-0.99). The MeltPro MTB assays had been used in 4 551 confirmed patients; the proportion of patients who obtained effective drug resistance results increased from 83.3% to 87.8%(P<0.01); and detection rate of rifampicin, isoniazid, ethambutol, fluoroquinolone resistance, multidrug and pre-extensive drug resistance cases were increased by 3.2%, 14.7%, 22.2%, 13.7%, 11.2% and 12.5%, respectively. Conclusion: The MeltPro MTB assays show satisfactory accuracy in the diagnosis of drug-resistant tuberculosis. This molecular pathological test is an effective complementary method in improving test positivity of drug-resistant tuberculosis.
Humans ; Rifampin/therapeutic use* ; Antibiotics, Antitubercular/therapeutic use* ; Mycobacterium tuberculosis ; Ethambutol/pharmacology* ; Isoniazid/pharmacology* ; Paraffin Embedding ; Retrospective Studies ; Cross-Sectional Studies ; Drug Resistance, Bacterial ; Sensitivity and Specificity ; Tuberculosis, Multidrug-Resistant/drug therapy*

Humans ; Floods ; Paraffin ; Paraffin Embedding

Humans ; Tuberculosis, Extrapulmonary ; Retrospective Studies ; Paraffin Embedding ; Tuberculosis/diagnosis* ; Mycobacterium tuberculosis/genetics* ; Formaldehyde ; Sensitivity and Specificity ; Sputum

OBJECTIVE: To optimize the method for embedding multiple undecalcified mouse tibias in plastic blocks, improve the efficiency and stability of plastic embedding and reduce the detachment rate of plastic slides. METHODS: Thirty undecalcified tibias from 15 B6 mice were used for plastic embedding after calcein labeling, fixation, dehydration and infiltration. The tibias were embedded in cylindrical plastic blocks with a diameter of 4 mm. For each bone, the 1/4 proximal tibia was cut off, and the remaining 3/4 was used for re-embedding. Five bones were embedded in a single block with each bone standing closely on the surface of a flat plate. The samples were randomized into control and experimental groups in all the processes of embedding, sectioning and staining. In the 3 groups with modified embedment, flowing CO was added into the embedding solution, embedding solution was applied to the section surface, and the slides were heated at 95 ℃ for 15 min. The polymerization time, slide detachment rate, bone formation and osteoblast parameters were analyzed. RESULTS: We prepared 6 plastic blocks, each containing 5 tibias, whose cross sections were on the same plane. The blocks were completely polymerized and suitable for sectioning. Flowing CO into the embedding solution reduced the polymerization time and increased the rate of complete polymerization. Application of the embedding solution on the section surface significantly reduced the detachment rate of the sections ( < 0.05) without affecting bone formation analysis ( > 0.05). Heating the slides significantly lowered the detachment rate of the sections ( < 0.05) without affecting osteoblast analysis ( > 0.05). CONCLUSIONS The optimized method allows effective embedding of multiple undecalcified mice tibias in the same block and can be an ideal method for histological analysis of undecalcified bones.
Animals ; Mice ; Plastics ; Staining and Labeling ; Tibia ; Tissue Embedding ; methods

Objective Quantitative analysis and comparison of the expression of ribonucleic acid （RNA） from frozen organs and formaldehyde-fixed and paraffin-embedded （FFPE） tissues. Methods Frozen specimens of human brain, myocardium and liver tissues as well as FFPE samples at different postmortem intervals were collected and mass concentration of RNA was extracted and detected. Reverse transcription-quantitative polymerase chain reaction （RT-qPCR） technology was used to analyze the amplification efficiency and relative expression of each RNA marker. Results The mass concentration and integrity of RNA extracted from FFPE samples were relatively low compared with frozen specimens. The amplification efficiency of RNA markers was related with RNA species and the length of amplification products. Among them, glyceraldehyde-3-phosphate dehydrogenase （GAPDH） and β-actin （ACTB） with relatively long amplification products failed to achieve optimal amplification efficiency, whereas 5S ribosomal RNA （5S rRNA） achieved ideal amplification efficiency and showed quite stable expression across various tissues, therefore it was chosen as internal reference marker. The expression quantity of GAPDH and ACTB in frozen specimens with longer postmortem intervals and in FFPE samples with relatively long amplification products was decreased. The expressions of tissue-specific microRNAs （miRNAs）, GAPDH and ACTB with relatively short amplification products had consistency in the same tissues and FFPE samples. Conclusion Through standardizing the RT-qPCR experiment, selecting the appropriate RNA marker and designing primers of appropriate product length, RNA expression levels of FFPE samples can be accurately quantified.
DNA Primers ; Formaldehyde ; Gene Expression Profiling ; Humans ; MicroRNAs/analysis* ; Myocardium ; Paraffin Embedding ; RNA/analysis* ; Real-Time Polymerase Chain Reaction/standards*

The role of androgens in the development of cardiovascular diseases remains controversial. The current study therefore sought to determine the changes in the histomorphology of the common carotid artery of the male rat in orchidectomy-induced hypogonadism. Twenty-two Rattus norvegicus male rats aged 2 months were used. The rats were randomly assigned into baseline (n=4), experimental (n=9), and control (n=9) groups. Hypogonadism was surgically induced in the experimental group by bilateral orchiectomy under local anesthesia. At experiment weeks 3, 6, and 9, three rats from each group (experimental and control) were euthanized, their common carotid artery harvested, and routine processing was done for paraffin embedding, sectioning, and staining. The photomicrographs were taken using a digital photomicroscope for morphometric analysis. Orchidectomy resulted in the development of vascular fibrosis, with a significant increase in collagen fiber density and decrease in smooth muscle and elastic fiber density. Moreover, there was development of intimal hyperplasia, with fragmentation of medial elastic lamellae in the common carotid artery of the castrated rats. Orchidectomy induces adverse changes in structure of the common carotid artery of the male rat. These changes may impair vascular function, therefore constituting a possible structural basis for the higher incidences of cardiovascular diseases observed in hypogonadism.
Androgens ; Anesthesia, Local ; Animals ; Cardiovascular Diseases ; Carotid Artery, Common* ; Collagen ; Elastic Tissue ; Fibrosis ; Humans ; Hyperplasia ; Hypogonadism* ; Incidence ; Male* ; Muscle, Smooth ; Orchiectomy ; Paraffin Embedding ; Rats*

Biomarkers, Tumor ; metabolism ; Formaldehyde ; Humans ; Neoplasms ; metabolism ; Paraffin Embedding ; RNA, Messenger ; metabolism ; Tissue Fixation

OBJECTIVETo investigate the frequency of anaplastic lymphoma kinase (ALK) expression in non-small cell lung cancer (NSCLC) patients and its correlation with the clinicopathologic features.

METHODSALK immunohistochemistry and ALK fluorescent in situ hybridization (FISH) were performed on formalin-fixed, paraffin-embedded tissue in 100 cases of NSCLCs between 2011 and 2013. Relevant clinicopathologic data were collected and correlated with ALK expression.

RESULTSAll patients with immunohistochemical score of 3 (n = 12) were FISH-positive and all patients with score of 0 (n = 78) were FISH-negative. Among patients with immunohistochemical scores of 1 and 2, 2/3 and 6/7 were FISH-positive, respectively. The sensitivity and specificity of ALK immunohistochemistry with intensity score of 1 or more were 100% and 98%, respectively. Invasive mucinous adenocarcinoma, solid or acinar growth pattern, presence of mucous cells (signet-ring cells or goblet cells), extracellular mucus and lack of significant nuclear pleomorphism characterized ALK-rearranged cancer.

CONCLUSIONSALK-rearranged cancers possess specific histological features. Immunohistochemistry can be used as a routine test for screening ALK-positive cases in advanced NSCLC, and FISH testing should be used to confirm ALK translocation for patients with tumors showing staining for ALK by immunohistochemistry. All of these can help physicians identify patients who may benefit from targeted therapy.

Carcinoma, Non-Small-Cell Lung ; enzymology ; Female ; Humans ; Immunohistochemistry ; In Situ Hybridization, Fluorescence ; Lung Neoplasms ; enzymology ; Male ; Paraffin Embedding ; Receptor Protein-Tyrosine Kinases ; metabolism ; Sensitivity and Specificity

OBJECTIVETo explore the feasibility of testing HER-2 expression and gene amplification in fine needle aspiration specimens of advanced breast cancers, and to benefit the patients receiving targeted drug therapy.

METHODSLiquid-based cytology specimens by fine needle aspiration of 49 breast cancer cases were used in this study. The expression of HER-2 protein was detected by immunocytochemistry (ICC) and the gene amplification was assessed by fluorescence in situ hybridization (FISH). All the 49 cases had overexpression of HER-2 protein marked as ++ or +++ in immunohistochemistry (IHC), and had corresponding FISH results in formalin-fixed, paraffin-embedded (FFPE) tissue samples.

RESULTSFNA samples in all the 49 cases were tested by FISH, and showed a complete agreement with the FISH results in the histological specimens (kappa = 1.0). Of the 49 cases, 33 had HER-2 gene amplification in FFPE samples. So do that in FNA samples. Both had an amplification rate of 67.3%. Among the 33 cases with HER-2 gene amplification, 26 had an ICC score of +++ (78.8%). The conformity rate was 78.8%. Of the 33 cases, 29 had an IHC score of +++ (87.9%). Its conformity rate was 87.9%. The difference between the ICC and IHC results was statistically not significant (P = 0.322). Among the 16 cases with negative gene amplification by both ICC and IHC, 15 cases showed HER-2 protein expression as 0/+, and another one case was not counted because there was not enough cells. Of the 16 cases, 15 had an IHC score of ++ and one of +++ . To take the FISH results as gold standard, ICC results had a high sensitivity (87.9%) and specificity (100.0%).

CONCLUSIONSFISH in FNA samples can be used in the clinic to test HER-2 gene amplification and overexpression in breast cancers, with a high sensitivity and specificity in ICC. Our data support the use of FISH and ICC analysis to determine HER-2 status on FNA specimens in patients with advanced breast cancer and recurrence or metastatic tumors. When ICC score is +++ , it indicates that there is a HER-2 gene amplification by FISH.

Adult ; Aged ; Biopsy, Fine-Needle ; Breast Neoplasms ; genetics ; metabolism ; pathology ; Carcinoma, Ductal, Breast ; genetics ; metabolism ; pathology ; Female ; Gene Amplification ; Humans ; Immunohistochemistry ; In Situ Hybridization, Fluorescence ; Lymphatic Metastasis ; Middle Aged ; Neoplasm Recurrence, Local ; Neoplasm Staging ; Paraffin Embedding ; Receptor, ErbB-2 ; genetics ; metabolism ; Sensitivity and Specificity

BACKGROUND: Inevitable loss of diagnostic material should be minimized during cell block preparation. We introduce a modified agarose cell block technique that enables the synthesis of compact cell blocks by using the entirety of a cell pellet without the loss of diagnostic material during cell block preparations. The feasibility of this technique is illustrated by high-throughput immunocytochemistry using high-density cell block microarray (CMA). METHODS: The cell pellets of Sure- Path residues were pre-embedded in ultra-low gelling temperature agarose gel and re-embedded in standard agarose gel. They were fixed, processed, and embedded in paraffin using the same method as tissue sample processing. The resulting agarose cell blocks were trimmed and represented on a CMA for high-throughput analysis using immunocytochemical staining. RESULTS: The SurePath residues were effectively and entirely incorporated into compact agarose cell buttons and embedded in paraffin. Sections of the agarose cell blocks revealed cellularities that correlated well with corresponding SurePath smears and had immunocytochemical features that were sufficient for diagnosis of difficult cases. CONCLUSIONS: This agarose-based compact cell block technique enables preparation of high-quality cell blocks by using up the residual SurePath samples without loss of diagnostic material during cell block preparation.
Biopsy, Fine-Needle ; Diagnosis ; Immunohistochemistry ; Paraffin ; Paraffin Embedding ; Sepharose*