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BACKGROUND: Infection is a leading cause of morbidity and death in patients with multiple myeloma (MM). The increased susceptibility to infection is complicated and multifactorial. However, no studies have explored the spectrum and risk factors of infections in newly diagnosed MM patients at the first admission. This cross-sectional study aimed to provide ideas for the assessment, prevention and treatment of infection in newly diagnosed MM patients when admitted for the first time. METHODS: Retrospectively, the data from electronic medical records for 161 patients newly diagnosed with MM from May 2013 to December 2018 were analysed. All the information was collected at the time of admission, and the patients had received no antineoplastic therapy previously. Independent risk factors of infection in multiple myeloma were determined by univariate and multivariate analysis. RESULTS: Newly diagnosed patients with MM were highly susceptible to viruses (43.9%), especially Epstein-Barr virus (EBV) (24.4%) and hepatitis B virus (HBV) (17.1%). Advanced stage (ISS stage III, P = 0.040), more severe anaemia (Hb < 90 g/L, P = 0.044) and elevated CRP (> 10 mg/L, P = 0.006) were independent risk factors for infection. Moreover, infections represented a major survival threat to patients with newly diagnosed MM (P = 0.033), and the existence of risk factors for infection was significantly correlated with poor prognosis (P = 0.011), especially ISS stage III (P = 0.008) and lower haemoglobin level (P = 0.039). CONCLUSIONS: Newly diagnosed MM patients are highly susceptible to viruses. Advanced ISS stage, more severe anaemia and the elevation of CRP are independent risk factors of infection, which also have a strong impact on prognosis. Our results suggest that viral infection should be taken into account if antibacterial drugs are not effective, and the prevention of infection and improvement of prognosis should be paid more attention in newly diagnosed patents with advanced stage and more severe anaemia.
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Infecções/etiologia , Mieloma Múltiplo/complicações , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos Transversais , Suscetibilidade a Doenças , Infecções por Vírus Epstein-Barr/etiologia , Feminino , Hepatite B/etiologia , Humanos , Infecções/mortalidade , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/mortalidade , Prognóstico , Estudos Retrospectivos , Fatores de RiscoRESUMO
Objectives In December 2019, a novel coronavirus (SARS-CoV-2)-infected pneumonia (COVID-19) occurred in Wuhan, China. Laboratory-based diagnostic tests utilized real-time reverse transcriptase polymerase chain reaction (RT-PCR) on throat samples. This study evaluated the diagnostic value to analyzing throat and sputum samples in order to improve accuracy and detection efficiency. Methods Paired specimens of throat swabs and sputum were obtained from 54 cases, and RNA was extracted and tested for 2019-nCoV (equated with SARS-CoV-2) by the RT-PCR assay. Results The positive rates of 2019-nCoV from sputum specimens and throat swabs were 76.9% and 44.2%, respectively. Sputum specimens showed a significantly higher positive rate than throat swabs in detecting viral nucleic acid using the RT-PCR assay (p = 0.001). Conclusions The detection rates of 2019-nCoV from sputum specimens were significantly higher than those from throat swabs. We suggest that sputum would benefit for the detection of 2019-nCoV in patients who produce sputum. The results can facilitate the selection of specimens and increase the accuracy of diagnosis.
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Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/genética , Pneumonia Viral/diagnóstico , Pneumonia Viral/genética , Manejo de Espécimes/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Betacoronavirus/genética , Betacoronavirus/patogenicidade , COVID-19 , China , Técnicas de Laboratório Clínico/métodos , Coronavirus/genética , Coronavirus/patogenicidade , Testes Diagnósticos de Rotina/métodos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pandemias , Faringe/virologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/patogenicidade , SARS-CoV-2 , EscarroRESUMO
BACKGROUND: In December 2019, a novel coronavirus (SARS-CoV-2) causing symptomatic illness (COVID-19) occurred in Wuhan, China. Travel-associated cases were reported in many other countries leading to epidemic transmission. The number of cases has increased rapidly but laboratory diagnosis is limited. METHODS: We collected samples from two groups of patients diagnosed with COVID-19 for experiments. In one group, 63 serum samples were analyzed IgG and IgM antibodies by enzyme-linked immunosorbent assay (ELISA) and 35 healthy serum samples were served as controls. In the other group, 91 plasma samples were analyzed by colloidal gold-immunochromatographic assay (GICA) for IgG and IgM antibodies and 35 healthy plasma samples were served as controls. Throat swab samples for nucleic acids retest were collected from 81/91 of these participant. RESULTS: The sensitivity of the combined ELISA IgM and IgG detection was 55/63 (87.3%). Sensitivity of the com-bined GICA IgM and IgG detection was 75/91 (82.4%). Both methods were negative for healthy controls and had a specificity of 100%. In 81 cases, the follow up throat swab samples were retested by RT-PCR, showing that 42 cases were positive. The sensitivity was 51.9% (42/81). The area under the receiver operating characteristic (ROC) curve for IgG (AUC(IgG)) was 0.934. The area under the ROC curve for IgM (AUC(IgM)) was 0.812. The area under the ROC curve for IgG + IgM (AUC(IgG+IgM)) was 0.983. CONCLUSIONS: The serological test of SARS-CoV-2 can be used as an important supplement to the existing RT-PCR test for the specific and rapid diagnosis of COVID-19. AUC(IgG) > AUC(IgM) indicates that IgG has better classification performance than IgM. AUC(IgG + IgM) > AUC(IgG) indicates that the combination of IgG and IgM has better classification performance than IgG alone.
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Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Imunoglobulina G/análise , Imunoglobulina M/análise , Pneumonia Viral/diagnóstico , Testes Sorológicos/métodos , Betacoronavirus/imunologia , COVID-19 , Teste para COVID-19 , Estudos de Casos e Controles , Infecções por Coronavirus/sangue , Infecções por Coronavirus/imunologia , Humanos , Pandemias , Pneumonia Viral/sangue , Pneumonia Viral/imunologia , SARS-CoV-2 , Sensibilidade e EspecificidadeRESUMO
BACKGROUND This retrospective study aimed to describe the effects of convalescent plasma therapy in 24 patients diagnosed with coronavirus disease 2019 (COVID-19) pneumonia due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection during February and March 2020 in Wuhan, China. MATERIAL AND METHODS The confirmation of SARS-CoV-2 infection was made by the reverse transcription-polymerase chain reaction test. We retrospectively analyzed the clinical data and laboratory test reports of patients with severe COVID-19 pneumonia who received a convalescent plasma transfusion. RESULTS A total of 24 patients with COVID-19 pneumonia who were transfused with ABO-compatible convalescent plasma were enrolled in the study. Convalescent plasma transfusion showed an effective clinical outcome in 14 of 24 patients (an effective rate of 58.3%). No patients had an adverse reaction to the transfusion. Compared with before convalescent plasma transfusion, the lymphocyte count after convalescent plasma transfusion increased to a normal level (median: 0.80×109/L vs. 1.12×109/L, P=0.004). Other laboratory indicators such as white blood cells, high-sensitivity C-reactive protein, procalcitonin, alanine aminotransferase, and aspartate transaminase showed a decreasing trend after transfusion. CONCLUSIONS This retrospective observational clinical study showed that convalescent plasma therapy could have beneficial effects on patient outcomes. Recently, regulatory authorization has been given for the use of convalescent plasma therapy, and clinical guidelines have been developed for the collection and use of convalescent plasma and hyperimmune immunoglobulin in patients with COVID-19.
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Transfusão de Componentes Sanguíneos/métodos , COVID-19/terapia , SARS-CoV-2/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , COVID-19/diagnóstico , COVID-19/imunologia , COVID-19/virologia , Teste de Ácido Nucleico para COVID-19 , China , Feminino , Humanos , Imunização Passiva/métodos , Masculino , Pessoa de Meia-Idade , RNA Viral/isolamento & purificação , Estudos Retrospectivos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Índice de Gravidade de Doença , Resultado do Tratamento , Soroterapia para COVID-19RESUMO
BACKGROUND: Numerous studies have demonstrated that Absent in Melanoma 2 (AIM2) is upregulated in aortic plaques, especially in Vascular Smooth Muscle Cells in Coronary Artery Disease (CAD), and is related to inflammasome-induced inflammation. However, the underlying mechanism of this phenomenon and the role of AIM2 in atherosclerosis remained unclear. METHODS: This study enrolled 133 CAD patients and 123 controls. We isolated Peripheral Blood Leukocytes (PBLs) and the mRNA expression of AIM2 inflammasome and its downstream genes (ASC, Caspase-1, IL-1ß, and IL-18) were detected by real-time quantitative PCR (qPCR). We assessed correlations between AIM2 expressions and clinical characteristics by multiple linear regression and spearman's correlation. The THP-1 cells cultured in poly(dA:dT), A151, interferon-gamma (IFN-γ), AG490, or JC2-11. And then the mRNA and protein levels of AIM2, ASC, Caspase-1, IL-1ß, IL-18, GSDMD, and STAT1 were analyzed by qPCR and Western blot analysis, respectively. The migration and adhesive capacity of THP-1 cells was assessed using an inverted microscope and an inverted fluorescence microscope, respectively. RESULTS: In this study, we found that expressions of components of AIM2 inflammasome and its downstream genes (ASC, Caspase-1, IL-1ß, and IL-18), were all increased in PBLs of CAD patients, which indicated the inflammasome activation. AIM2 inflammasome activation further induced pyroptosis, and stimulated migration and adhesion in monocyte cell lines, which was regulated by IFN-γ probably through JAK2/STAT1 pathway. In addition, AIM2 expressions were positively correlated with systemic inflammatory indicators as an independent risk factor for CAD. CONCLUSIONS: In conclusion, increased AIM2 expression, induced by the IFN-γ/JAK2/STAT1 signal, orientates monocytes to inflammatory status or even pyroptosis through AIM2 inflammasome activation, which is involved in the development of CAD.
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Doença da Artéria Coronariana , Proteínas de Ligação a DNA , Inflamassomos , Interferon gama , Janus Quinase 2 , Monócitos , Piroptose , Fator de Transcrição STAT1 , Transdução de Sinais , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Doença da Artéria Coronariana/imunologia , Doença da Artéria Coronariana/metabolismo , Doença da Artéria Coronariana/patologia , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Inflamassomos/metabolismo , Interferon gama/metabolismo , Janus Quinase 2/metabolismo , Janus Quinase 2/genética , Monócitos/metabolismo , Monócitos/imunologia , Fator de Transcrição STAT1/metabolismo , Células THP-1RESUMO
The proapoptotic caspase adaptor protein (PACAP) is involved in cell-cycle regulation and promotes apoptosis. Both MGC29506 and PACAP are isoforms of the MGC29506 gene and are generated by differential splicing of the alternative splice-acceptor. In studying PACAP, we inadvertently constructed the eukaryotic expression vector MGC29506. At present, the function of the MGC29506 gene is largely unknown with the key exception of information obtained by bioinformatics. We studied the role of MGC29506 in gastric cancer cell proliferation, the cell cycle and apoptosis. In addition, we studied MGC29506 expression in gastric cancer patients and explored its significance. We found that the expression of MGC29506 in gastric cancer samples was lower than in samples from adjacent non-tumor tissues. We found that the MGC29506 protein was localized in the cell nucleus of AGS cells and inhibited their proliferation. Higher percentages of G0/G1 and S phase cells were induced by transfection with the MGC29506 gene than were induced by transfection with the negative control. We showed that cells transfected with MGC29506 were arrested at the G0/G1 and S phases of the cell cycle. However, we found no significant increases in apoptosis of cells transfected with MGC29506 compared with cells transfected with the negative control. Our results suggested that MGC29506 has the potential of functioning as a novel suppressor gene in gastric cancer. Downregulation of MGC29506 may also promote the progression of gastric cancer.
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Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Pontos de Checagem do Ciclo Celular/fisiologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Neoplasias Gástricas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Idoso , Apoptose , Ciclo Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , Regulação para Baixo , Feminino , Humanos , Masculino , Neoplasias Gástricas/genéticaRESUMO
OBJECTIVE: To observe peripheral blood chromosome abnormality and microdeletions of the SRY and AZF genes on the Y chromosome in patients with chimera Klinefelter syndrome. METHODS: We analyzed the cytogenetic karyotype of the peripheral blood chromosome in 1 infertile patient with mosaic karyotype Klinefelter syndrome and his parents. We identified 9 sequence tagged sites (STS) by multiplex PCR: sY84, sY86, sY127, sY129, sY134, sY254, sY255, sY242, and sY152. Meanwhile we detected the SRYgene and the microdeletion of AZF using ZFX/ZFY as the internal control gene. RESULTS: The karyotype of the patient was 46,XY (12%)/47,XXY (30%)/48,XXYY (56%)/49,XXXXY (2%). The karyotypes of his parents were normal. Consistency was found between the SRY gene and the chromosome gender in the patient and his parents. Y chromosome AZF microdeletion was observed in the patient. The deletion sites were sY86 and sY127, and the deletion type was AZFa + AZFb. CONCLUSION: AZF microdeletion of the Y chromosome exists in patients with Klinefelter syndrome. Chromosome karyotype and Y-chromosome AZF microdeletion are important criteria for the genetic diagnosis of Klinefelter syndrome.
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Síndrome de Klinefelter/genética , Transtornos do Cromossomo Sexual no Desenvolvimento Sexual/genética , Quimerismo , Deleção Cromossômica , Cromossomos Humanos Y/genética , Citogenética , Humanos , Infertilidade Masculina , Cariotipagem , Masculino , Sitios de Sequências Rotuladas , Aberrações dos Cromossomos Sexuais , Adulto JovemRESUMO
Massive blood transfusion (MBT) is a relatively common complication of cardiac surgery, which is independently associated with severe postoperative adverse events. However, the value of using rapid thrombotomography (r-TEG) to predict MBT in perioperative period of cardiac surgery has not been explored. This study aimed to identify the effect of r-TEG in predicting MBT for patients undergoing coronary artery bypass grafting (CABG).This retrospective study included consecutive patients first time undergoing CABG at the Zhongnan Hospital of Wuhan University between March 2015 and November 2017. All the patients had done r-TEG tests before surgery. The MBT was defined as receiving at least 4 units of red blood cells intra-operatively and 5 units postoperatively (1 unit red blood cells from 200âmL whole blood).Lower preoperative hemoglobin level (Pâ=â.001) and longer cardiopulmonary bypass time (Pâ=â.001) were the independent risk factors for MBT during surgery, and no components of the r-TEG predicted MBT during surgery. Meanwhile, longer activated clotting time (Pâ<â.001), less autologous blood transfusion (Pâ=â.001), and older age (Pâ=â.008) were the independent risk factors for MBT within 24âhours of surgery.Preoperative r-TEG activated clotting time can predict the increase of postoperative MBT in patients undergoing CABG. We recommend the careful monitoring of coagulation system with r-TEG, which allows rapid diagnosis of coagulation abnormalities even before the start of surgery.
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Transtornos da Coagulação Sanguínea/diagnóstico por imagem , Testes de Coagulação Sanguínea/métodos , Ponte de Artéria Coronária/efeitos adversos , Doença da Artéria Coronariana/diagnóstico por imagem , Complicações Pós-Operatórias/diagnóstico por imagem , Tromboelastografia/métodos , Idoso , Transtornos da Coagulação Sanguínea/etiologia , Transfusão de Sangue/estatística & dados numéricos , Doença da Artéria Coronariana/cirurgia , Diagnóstico Precoce , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias/etiologia , Valor Preditivo dos Testes , Período Pré-Operatório , Estudos Retrospectivos , Fatores de Risco , Fatores de TempoRESUMO
BACKGROUND: Identification of RhD antigen epitopes is a key component in understanding the pathogenesis of haemolytic disease of the foetus and newborn. Research has indicated that phage display libraries are useful tools for identifying novel mimic epitopes (mimotopes) which may help to determine antigen specificity. MATERIALS AND METHODS: We selected the mimotopes of blood group RhD antigen by affinity panning a phage display library using monoclonal anti-D. After three rounds of biopanning, positive phage clones were identified by enzyme-linked immunosorbent assay (ELISA) and then sent for sequencing and peptides synthesis. Next, competitive ELISA and erythrocyte haemagglutination inhibition tests were carried out to confirm the inhibitory activity of the synthetic peptide. To evaluate the diagnostic performance of the synthetic peptide, a diagnostic ELISA was examined. RESULTS: Fourteen of 35 phage clones that were chosen randomly from the titering plate were considered to be positive. Following DNA sequencing and translation, 11 phage clones were found to represent the same peptide - RMKMLMMLMRRK (P4) - whereas each of the other three clones represented a unique peptide. Through the competitive ELISA and erythrocyte haemagglutination inhibition tests, the peptide (P4) was verified to have the ability to mimic the RhD antigen. The diagnostic ELISA for P4 proved to be sensitive (82.61%) and specific (88.57%). DISCUSSION: This study reveals that the P4 peptide can mimic RhD antigen and paves the way for the development of promising targeted diagnostic and therapeutic platforms for haemolytic disease of the foetus and newborn.
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Epitopos/química , Biblioteca de Peptídeos , Sistema do Grupo Sanguíneo Rh-Hr/química , Epitopos/genética , Humanos , Peptidomiméticos/química , Sistema do Grupo Sanguíneo Rh-Hr/genéticaRESUMO
OBJECTIVE: To establish a sensitive method for the early detection of circulating tumor cells (CTCs) in peripheral blood (PB) of colorectal cancer (CRC) patients. METHODS: PB samples were collected from 156 CRC patients, 40 benign colorectal disease patients, 40 healthy individuals and 45 patients with other solid tumors. The combination of negative and positive immunomagnetic bead method was used to enrich cancer cells. Then, cytokeratin-20 (CK20), survivin and carcinoembryonic antigen (CEA) mRNA were detected by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). In addition, analyses were carried out for their correlation with patients' clinicopathologic features. RESULTS: The positive rates of survivin, CK20 and CEA mRNA in the PB of CRC patients were 57.7, 47.4 and 39.1%, respectively, and the sensitivity increased from 39.1% of CEA mRNA alone to 60.9% of the combined panel. The expression of the three mRNAs in CRC patients was significantly higher than that in benign control and healthy volunteers, and the expression of survivin and CK20 was not significantly higher than that of patients with other solid tumors. However, the expression of CEA mRNA was significantly higher than that of patients with other solid tumors. The expression of survivin, CK20 and CEA mRNA was significantly correlated with Dukes stages and lymph node metastasis. CONCLUSIONS: The combined use of negative and positive immunomagnetic beads followed by amplification of survivin, CK20 and CEA mRNA by means of qRT-PCR is a non-invasive and sensitive assay for the detection of circulating CRC cells. The combined panel improved the sensitivity of detection in CRC patients.
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Antígeno Carcinoembrionário/sangue , Neoplasias Colorretais/sangue , Neoplasias Colorretais/diagnóstico , Proteínas Associadas aos Microtúbulos/sangue , Proteínas de Neoplasias/sangue , Células Neoplásicas Circulantes/química , RNA Mensageiro/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Bases , Antígeno Carcinoembrionário/genética , Linhagem Celular Tumoral , Feminino , Humanos , Proteínas Inibidoras de Apoptose , Queratina-20/sangue , Queratina-20/genética , Leucócitos Mononucleares/química , Leucócitos Mononucleares/citologia , Masculino , Proteínas Associadas aos Microtúbulos/genética , Pessoa de Meia-Idade , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SurvivinaRESUMO
BACKGROUND: Vascular endothelial growth factor (VEGF) is a key mediator that plays an important role in angiogenesis, tumor growth, and tumor metastasis. The associations between five polymorphisms of VEGF (rs3025039, rs699947, rs10434, rs1570360, and rs2010963) and renal cell carcinoma (RCC) risk have been extensively investigated, but the currently available results are inconsistent and inconclusive. To obtain a more accurate assessment of the associations, we conducted a meta-analysis in this study. MATERIALS AND METHODS: Relevant studies were collected systemically from the following three electronic databases: MEDLINE, Web of Science, and CNKI (Chinese National Knowledge Infrastructure). Statistical analyses were performed using Review Manager 5.2 in a fixed- or random-effects model. Pooled odds ratios (ORs) with 95% confidence intervals (95% CIs) were calculated to establish the strength of associations. RESULTS: A total of eight case-control studies with 1,936 RCC cases and 2,770 controls fulfilling the inclusion criteria were selected for this meta-analysis. The pooled OR indicated that rs699947 polymorphism was significantly associated with RCC risk in all genetic models. A significant association was also found between the rs3025039 polymorphism and RCC risk in a homozygous model (TT vs CC: OR =1.38, 95% CI =1.11-1.72, P=0.004), a dominant model (CT+TT vs CC: OR =1.21, 95% CI =1.05-1.39, P=0.01), and a recessive model (TT vs CC+CT: OR =1.28, 95% CI =1.04-1.57, P=0.02). After a subgroup analysis of ethnicity in the allele contrast model of rs3025039 polymorphism, we found a significant relationship in the allele contrast model (T vs C: OR =1.21, 95% CI =1.05-1.40, P=0.007) in the Asian population. With regard to rs10434 polymorphism, significant association was observed only in a homozygous model (GG vs AA: OR =0.75, 95% CI =0.57-0.98, P=0.03). As to rs1570360 or rs2010963, we did not observe any relationship between the two polymorphisms and RCC risk in our study. CONCLUSION: Our meta-analysis confirmed the fact that rs699947, rs3025039, and rs10434 polymorphisms were significantly relevant to elevated RCC risk. In the meanwhile, this study also demonstrated that the allele contrast model of rs3025039 polymorphism was likely to be associated with risk of RCC in the Asian population.
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OBJECTIVES: To develop a specific, reliable assay for detecting circulating tumor cells (CTC) in peripheral blood of breast cancer patients. DESIGN AND METHODS: 94 breast cancer patients, 35 patients with benign breast tumor, 40 healthy individuals, and 25 patients with other solid tumors were evaluated by quantitative real-time reverse transcription-PCR (qRT-PCR) for detecting Survivin, hTERT, and hMAM mRNA in peripheral blood (PB) of breast cancer patients. In addition, analyses were carried out for their correlation with patients' clinicopathologic features. RESULTS: The sensitivity of Survivin, hTERT, and hMAM mRNA in the PB of breast cancer patients was 36.2%, 59.6% and 33.0%, respectively. Survivin and hTERT were detected in the PB patients with solid tumors other than breast cancer, but hMAM mRNA was only detected in breast cancer patients. The sensitivity of three combined markers in the parallel test was 70.2%.Compared to that of single marker detection, the three combined markers' percentage was significantly higher. However, the specificity of three combined markers of serial test was 100%. The expression of the three mRNAs significantly correlated with TNM stage, and lymph node metastasis. CONCLUSIONS: Survivin, hTERT and hMAM mRNA assays are powerful methods for detection of CTC of breast cancer patients. With combination of the three markers for detection of CTC of breast cancer, the parallel test increases the sensitivity. This analysis can offer a simple, noninvasive, and promising adjuvant tool for the early detection of micrometastatic tumor cells in breast cancer patients.