Progress in molecular diagnostic techniques for leukemia / 中华检验医学杂志

With the development of molecular diagnostic techniques, the study for the etiology of leukemia has been entering the era of the molecular biology.Molecular techniques for leukemia diagnosis, prognosis and individualized therapy are used widely, becoming one of the necessary routine tests for patients with leukemia. So far, molecular techniques for leukemiaincluding cytogenetic diagnosis, molecular genetics, molecular diagnostics based on PCR, mutation detection, as well as a variety of next-generation sequencing, have played an important role in the diagnosis of leukemia, minimal residual disease monitoring, prognosis and targeted therapy.

Association of polymorphism in the promoter region of PCA3 gene with risk of prosate cancer / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the polymorphism in the promoter region of PCA3 gene and its relationship with risk of prostate cancer (PCa).</p><p><b>METHODS</b>The promoter region of PCA3 gene of the DNA of peripheral blood mononuclear cells was detected by sequence analysis in the 186 PCa and 141 BPH patients and 135 healthy control individuals. If the samples were detected with polymorphism of insection/deletion, clone sequence analysis was used with pBS-T carrier to verify it.</p><p><b>RESULTS</b>There were 5 polymorphisms. TAAA repeat times: 4, 5, 6, 7, 8, and 8 genotypes (TAAA 4/5, TAAA 4/6, TAAA 5/5, TAAA 5/6, TAAA 5/7, TAAA 5/8, TAAA 6/6, and TAAA 6/7) were detected in the promoter region of PCA3 gene. The eight genotypes were divided into three groups: ≤10TAAA, 11TAAA, ≥12TAAA. Unconditional logistic regression analysis models were used to analyze the relationship between different genotypes and cancer risks adjusted by sex and age. The type 11TAAA and ≥12TAAA was associated with higher relative risk for prostate cancer than the group ≤10TAAA [OR=1.74, 95% CI=1.06-2.87 (for type 11TAAA); OR=5.63, 95% CI=1.85-17.19 (for type ≥12TAAA)]. In the 186 PCa patients, there was 62.4% allele of PCA3 gene with AG/CA mutation found in the promoter 18-19 bp region of PCA3 gene and it had a close relation with the development of prostate cancer.</p><p><b>CONCLUSIONS</b>Short tandem repeats are found in the promoter region of the PCA3 gene in PCa patients, and the increase of TAAA repeat sequences highly enhance the relative risk of prostate cancer development. The occurrence of such STR might be related to the mutations in their upstream loci.</p>

Pyrosequencing for detection of drug resistant relevant mutation in the polymerase gene of Hepatitis B virus and its clinical application / 中华检验医学杂志

Objective To explore the accuracy and clinical application of Pyrosequencing for detection of drug resistant relevant mutation in the polymerase gene of Hepatitis B virus (HBV).Methods Compared with Sanger sequencing,the accuracy and sensitivity of Pyrosequencing were assessed.Pyrosequencing was used to determine the serum of 1164 patients with chronic Hepatitis B and its results were analyzed.Results The sensitivity of Pyrosequencing was 1 × 103 KIU/L,the same as Sanger sequencing.But Pyrosequencing was more stable and specific than Sanger sequencing to detect low rate of mutation with over 10％ mutation.The mutations couldn't be detected in 248 patients with chronic Hepatitis B,but positive results occurred in 919 patients,among them,61.5 ％ without mutation,38.5 ％ with mutation (including 47.5％ of the single locus mutation and 52.5％ of the combined mutation).Other sites had different degree of mutations except the sites of rtI169T and rtA194T; the main mutation sites in patients with chronic Hepatitis B were rtM204V/I (32.1％),rtL180M (19.8％),rtA181V/T (6.7％) and rtN236T (5.3％),in which fractional mutation had high ratio in the sites of rtA181V/T (6.7％) and rtN236T (5.3％).Conclusions Pyrosequencing has good sensitivity,specificity and accuracy and can early detect the drug resistant relevant mutation in the polymerase gene of HBV,which is suitable for monitoring patients with chronic Hepatitis B for the treatment of nucleoside analogues (acid).The different sites and frequencies of mutations may be associated with different habits of doctors for using different anti-HBV drugs.

Analysis of suspicious results of serum HBV DNA detected by fluorescence quantitative PCR / 中华检验医学杂志

Objective To analyze the suspicious results of serum HBV DNA by fluorescence quantitative PCR and develop appropriate countermeasures in order to improve the quality of detection of HBV DNA.Methods Blood samples of patients from the First Affiliated Hospital of Wenzhou Medical College from 2008 to 2011 were analyzed for HBV DNA by fluorescence quantitative PCR.1969 cases of suspicious results,judged by the rule of review the results of serum HBV DNA combined with the historical results,PCR amplification curve,HBV serum markers and clinical diagnosis,were analyzed and redetected by using of two different reagents,careHBV PCR Kit and careHBV PCR Kit V2,at the same time.The consistency and inconsistency ratio of the results were evaluated.Both the reasons of inconsistent and the undetected rates of careHBV PCR Kit were analyzed.The two reasons for the inconsistent results included the reagent related factors,e.g,showing no amplification curve caused by the false negative and abnormal low efficiency of amplification curve,and the non reagent related factors such as operating pollution and other sample factors.Results There were 115 154 blood samples were detected for HBV from 2008 to 2011 and 1969 samples (1.71％) with suspicious results were redetected.The consistency and inconsistency results were 1588 (80.65％) and 381 (19.35％),respectively.Every year from 2008 to 2011,the percentage of the inconsistent results caused by the reagent related factors were 18.87％,20.23％,51.33％ and 59.57％ respectively,which showed an increasing trend,and the percentage of inconsistent results caused by the nonreagent related factors were 81.13％,79.77％,48.67％ and 40.43％ respectively,which showed a declining trend year by year.The undetected rates of careHBV PCR Kit were 2.49％,4.08％,10.09％ and 14.47％ respectively,showing an increasing trend.Conclusions The redetection for the specimens with the suspicious results by using of different reagents can avoid the blind detection of HBV DNA and reduce the experimental error.All the clinical samples for quantitative HBV DNA including the mutations of HBV gene can be measured accurately and effectively,which is helpful to hepatitis B patients for antiviral therapy.

Significance of tyrosine kinase receptor gene and its variant expressions in bladder tumor tissues / 中华检验医学杂志

ObjectivesTo explore the clinical significance of tyrosine kinase receptor RON mRNA expression and it's splicing variant in bladder tumors. Methods Sixty-three cases of transitional cell carcinoma of the bladder (TCCB), including 30 cases of pathological grade I , 15 cases of pathological grade Ⅱ and 18 cases of pathological grade Ⅲ (44 cases of clinical stage Tis + T1, 15 cases of T2 + T3 +T4), 7 inverted papilloma of the bladder ( IPB), 9 cases of papillary urothelial neoplasm of low malignant potential (PUNLMP) and 12 cases of normalbladder mucosa RT-PCR was employed with the internal standard (GAPDHmRNA) to detect the expression of RON mRNA. PCR and direct sequencing was then utilized to identify the potential RON mRNA splicing variants. Finally, the variants' positive rates of expression were analyzed among the different tissues, diverse TCCB pathological grades and clinical stages. ResultsThe expression levels of RON mRNA/GAPDH mRNA among TCCB, IPB, PUNLMP and normal control were 4. 9 × 10-3 ( 1. 8 × 10-3-1.0 × 10-2 ), 3. 8 × 10-3 (2. 4 × 10-3-1.7 × 10-2 ), 4. 9 ×10 -3 ( 1.7 × 10 -3-1.1 × 10 -2 ) and 1.0 × 10-3 (4. 5 × 10-4-2. 8 × 10-3 ) respectively. The difference had a statistical significance (x2K-W = 17. 278 ,P ＜0. 05 ). The expression levels among pathological grade I, Ⅱ andⅢ were 3.7 × 10-3( 1.3 × 10-3-7.5 × 10-3) , 4. 9 × 10-3(1.9 × 10-3-1.1 × 10-2) and 8.9 × 10-3(2. 7 ×10 -3-8.0 × 10 -2 ) respectively. The erpression levels among the clinical stage Tis + T1 and T2 + T3 + T4were 3.5 × 10-3 ( 1.2 × 10 -3-7. 7 × 10-3 ) and 9. 7 × 10 -3 ( 2. 9 × 10-3-5. 3 × 10-2 ). The differences between expression levels were of statistical significance among the different pathological grades ( x2k-W =7. 341, P ＜0. 05 ) and clinical stages ( Z = - 2. 306, P ＜ 0. 05 ). The positive rates of exon 11deletion(E11△) in TCCB, IPB and PUNLMP were 71％ (45/63), 57％ (4/7) and 67％ (6/9) respectively, andthe total positive rate in bladder tumor tissues was 70％. Meanwhile, expression of the novel RON variant wesnot detected in the normalbladder mucosa. The positive expression rate of E1 1△ has no significant correlationamong the different clinical pathological tissues (x2 = 0. 620, P ＞ 0. 05 ). There was no statistical significancein expression positive rate between different pathological grades ( Z =0. 221, P ＞0. 05 ) and clinical stages( Z = 0. 538, P ＞ 0. 05) as well. A novel RON splice variant, deletion of RON exon 11 3 476 - 3 539 ( E3476 -3539△) was fond in the pathological tissue. The positive expression rates of E3 476 -3 539 in TCCB,IPB and PUNLMP were 57％ (36/63), 43％ (3/7) and 56％ (5/9) respectively, and the total positive expression rate was 56％ (44/79). The positive rates of E3 476 -3 539△ in pathological grade I , Ⅱ and Ⅲ were 40％ ( 12/30), 67％ (10/15) and 78％ (14/18), and it's positive rates in clinical stage Tis +T1and T2 +T3 + T4 were 48％ (21/44) and 80％ (12/15). The differences in each group had significantly statistical significance ( Z = 7. 285, 5. 041, P ＜ 0. 05 ) . However, the positive rates amongdifferent pathological tissues had no significance (x2 = 0. 517, P ＞ 0. 05 ). Conclusions The expression level of RON mRNA is significantly associated with histological grading and clinical stage. RON may play an important role in the progression ofTCCB. Compared with the normal control, the increased RON variant expression may contribute to the carcinogenesis of the bladder tumor.

Prevalence of human papillomavirus infection in women with uterine cervical cancer in Wenzhou / 中华临床感染病杂志

Objective To investigate the prevalence of human papillomavirus(HPV)infections in women with uterine cervical cancer in Wenzhou.Methods Exfoliated cells samples of cervix uteri were collected from 198 patients with cervical cancer. Flow-through hybridization technique was used to detect HPV and its genotypes.The relationship of HPV infection with cervical cancer stage,histological type and differentiation degree were analyzed.The prevalence of HPV infections in patients with different cervical diseases was observed.SPSS 13.0 was used for statistical analysis.Results In 198 patients with cervical cancer,HPV infection was occunrred in 147 (74.24%), of whom 101patients were superinfected (51.01%),and 129 patients(65.15%)were infected with the high-risk HPVs,which were significantly higher than those in cervicitis and cervical dysplasia(x2 = 28.28,65.34 and 95.22,P ＜ 0.01).HPV positive rate was not correlated with clinical stages,differentiation degree of cervical cancer(x2 = 0.475 and 0.969,P＞0.05).HPV positive rates in squamous cancer and adenocarcinoma had no statistical difference (x2 =0.582,P＞0.05).The logistic regression analysis showed that HPV 16/58 infection and age over 40might increase the risk of carcinogenesis of the cervix.Conclusion sHPV infection and superinfection are popular in women with cervix cancer in Wenzhou.HPV16/58 infection and age over 40 years are the risk factors of cervical cancer.

Detection of aberrant methylation of RARβ2, GSTP1 and DAPK gene in prostate cancer and its clinical significance / 中华老年医学杂志

Objective To explore hypermethylation of RARβ2, GSTP1 and DAPK gene in prostate cancer tissues, and to explore its correlation with clinicopathological features of prostate cancer and its diagnostic value. Methods Hypermethylation of RARe2, GSTP1 and DAPK gene was detected by nested methylation-specific PCR (NMSP) in 57 prostate cancer (PCa) tissues and 35 benign prostate hyperplasia (BPH) tissues. The correlation between hypermethylation and clinicopathological features of prostate cancer and its diagnostic value were analyzed. Results The hypermethylation frequencies of RARβ2, GSTP1 and DAPK gene in PCa were significantly higher than those in BPH (RARβ2: 52.6% vs. 0% GSTP1: 61.4% vs. 2.9%;DAPK: 43.9% vs. 8.6%;all P<0.01). The methylation rate of RARβ2 gene was directly correlated with distinct Gleason scores and clinical stage (4～7 score vs. 8～10 score: 34.8% vs. 64.7%;stage B, C vs. stage D: 37.0% vs. 66.7%;x2=4.927 and 5.004, P=0.026 and 0.025). The methylation rate of GSTP1 gene was significantly different in patients with different Gleason scores (4～7 score vs. 8～10 score: 43.5% vs. 73.5%;x2 =11.530, P=0.001), but had no difference in patients with distinct clinical stage (stage B, C vs. stage D: 51.9% vs. 70.0%;x2=1.975, P=0.16) . There was no difference in DAPK gene methylation rate among patients with different Gleason scores and distinct clinical stage (4 ～7 score vs. 8～10 score: 39.1% vs. 50.0%;stage B, C vs. stage D: 33.3% vs. 53.3%;x2= 1.290 and 2.309, both P～0.05). GSTP1 gene showed the highest sensitivity of 61.4% (35/57)with specificity of 97.1%(34/35), while RARβ2 gene had the highest specificity of 100% (35/35) with the sensitivity of 52.6% (30/57). The sensitivity and specificity of DAPK gene were 43.9% and 91.4% (25/57 and 32/35), respeetively. When the hypermethylation of RARβ2, GSTP1 and DAPK gene were detected together, the diagnostic sensitivity was increased, but the specificity was decreased. Conclusions The aberrant methylation of RARβ2, GSTP1 and DAPK gene is correlated with tumorigenesis and progression of prostate cancer, which may be used as an effective diagnostic marker for prostate cancer.

The value of the ratio of transmembrane protease serine 2 and ETS-related gene fusion transcripts in the diagnosis of prostate cancer / 中华检验医学杂志

n be used as a potential marker in the diagnosis of PCa. However, it can not be used as an index to monitor tumor progression or prognosis in PCa patients.

Flow-through Hybridization and Its Clinical Application to Human Papillomavirus Subtype Detection / 中华医院感染学杂志

OBJECTIVE To evaluate the clinical application of flow-through hybridization to human papillomavirus(HPV) subtype detection.METHODS A total of 305 female patients infected with HPV were selected for HPV subtype analysis using flow-through hybridization and the assessment of HPV subtype on cervical lesions was analyzed.RESULTS All 21 different subtypes were found.From 305 cases 173 patients were infected with single-type HPV and 132 patients were infected with multiple-type HPV.The most commonly found high-risk types were HPV16(17.6%),HPV52(9.8%),HPV58(9.0%),HPV33(6.3%),HPV18(5.4%) and HPV68(5.2%) and the low-risk types were HPV11(14.3%) and HPV6(6.5%).The high-risk HPV increased and the low-risk HPV declined along with the upgrade of the cervical lesions.CONCLUSIONS Technology of flow-through hybridization is able to detect multiple HPV subtype.The distinction between low and high-risk HPV subtypes is seemed useful in prevention and management of cervical cancer.

Quantitative detection of DD3 mRNA and PSA mRNA expressions in prostate cancer tissues by realtime fluorescent quantitative reverse transcription polymerase chain reaction / 中华泌尿外科杂志

Objective To study the expressions of DD3 mRNA and PSA mRNA in the prostate tissues and its diagnostic value in prostate cancer (PCa). Methods DD3 mRNA and PSA mRNA were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction ( FQ-RT-PCR) based on Taqman technique in the tissues of 21 cases of PCa and 39 cases of BPH. The receiver operating characteristic (ROC) curve was used to evaluate the diagnostic efficacy of DD3 mRNA, PSA mRNA and DD3 mRNA/PSA mRNA. Results The expressions of DD3 mRNA and PSA mRNA, and DD3 mRNA/ PSA mRNA were significantly higher in PCa tissues than those in BPH tissues ( P 0.05 for all). The AUC-ROC of DD3 mRNA,PSA mRNA and DD3 mRNA/PSA mRNA were 0. 937 (95% CI,0. 879 -0. 995) , 0.755(95% CI,0.629 -0.880) and 0.839 (95%CI,0.738 -0.940),respectively. The sensitivity for DD3 mRNA,PSA mRNA and DD3 mRNA/PSA mRNA was 90. 5% ,81. 0% and 81. 0% , respectively, and the specificity was 85.0% ,62.0% and 66.7% at cutoff value of 1.4?105 copies/mg tissue,3.0?107 copies/ mg tissue and 5. 0?10-3,respectively. The sensitivity and specificity of simultaneous detection for DD3 mRNA and PSA mRNA were 100% and 85.0%. Conclusions Both DD3 mRNA and PSA mRNA expressions were significantly higher in PCa tissues than those in BPH tissues; and the quantitative detection of DD3 mRNA is more helpful for the diagnosis. The simultaneous detection of DD3 mRNA and PSA mRNA can improve the sensitivity in the diagnosis of PCa.

RP-HPLC fingerprint chromatogram of Baohe Pill / 中成药

AIM: To provide a basis for appraising quality standard of Baohe Pill (Fructus Gataegi, Massa medicata fermentata, Rhizoma pinelliae, Pericapium citr reticulatae, Fructus forsythiae, Semen raphani, Fructus Mordei germinatus, Poria) inclusive of its RP-HPLC fingerprint chromatogram. METHODS: The chromatographic column was Kromasil C_ 18 column (4.6 mm?250 mm, i.d, 5-?m particle size). The mobile phase was 0.5% solution of ammonium dihydrogen phosphate (NH_4H_2PO_4), and the flow rate was 0.8 mL/min with UV detector at 214 nm. RESULTS: The RP-HPLC fingerprint chromatogram of Baohe Pill was established. In the experiment, for precision and repeatability, the RSD of each area of common peak was less than 3%. CONCLUSION: This method is simple and reliable.