Metabolomic Analysis of Urine in Rat Model with Spleen-stomach Damp-heat Syndrome / 中国实验方剂学杂志

ObjectiveTaking the rat model of spleen-stomach damp-heat syndrome（SSDHS） as the research object， this study aimed to investigate the potential biomarkers of SSDHS and the related metabolic pathways based on urine metabolomics， and tried to reveal the essence of SSDHS at the level of endogenous small molecular metabolites. MethodSixteen SD rats were randomly divided into normal and model groups. The normal group was fed normal chow and the model group was fed with 200 g·L-1 honey water daily， and lard and Chinese Baijiu alternately on alternate days for 17 days. The SSDHS model rats were exposed to external dampness-heat environment with temperature at 30-34 ℃， relative humidity of 95% for 2 h at the same time every day from the 10th day for 7 d. Then， the model was evaluated by observing the general conditions of the rats， measuring the contents of motilin（MTL） and gastrin（GT） in plasma by enzyme-linked immunosorbent assay（ELISA）， and examining the histopathology of gastronitestinal tissues. In additon， the urine metabolomics analysis was performed by ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS）， and the detection conditions was as follows：ACQUITY™ UPLC BEH C18 column（2.1 mm×100 mm， 1.7 μm）， mobile phase of 0.1% formic acid aqueous solution（A）-0.1% formic acid acetonitrile solution（B） for gradient elution （0-3 min， 1%-18%B； 3-8 min， 18%-40%B； 8-10 min， 40%-100%B）， the flow rate of 0.4 mL·min-1， electrospray ionization（ESI） in positive and negative ion modes， scanning range of m/z 50-1 000. The univariate and multivariate statistical analysis were constructed for screening inter-group differential ions， the element composition was calculated according to the precise relative molecular weight， and ion information was matched with databases such as Human Metabolome Database（HMDB） to identify biomarkers. Kyoto Encyclopedia of Genes and Genomes（KEGG） database was used to obtain the biological information of metabolites， and their associated metabolic pathways were analyzed by MetaboAnalyst 5.0. ResultCompared with the normal group， the rectal temperature of the model group increased significantly（P<0.01）， the levels of plasma MTL and GT decreased significantly（P<0.05， P<0.01）， and pathological changes such as bleeding， congestion and inflammatory infiltration in the gastric and colonic tissues. A total of 25 differential metabolites such as L-histidine， citric acid and isocitric acid were found to be the potential biomarker of SSDHS by urine metabolomics， 13 of which were phase Ⅱ metabolites of endogenous substances（glucuronic acid conjugates， sulfuric acid conjugates and acetyl conjugates）， involving the metabolic pathways of histidine metabolism， tricarboxylic acid cycle， glyoxylate and dicarboxylate metabolism. ConclusionSSDHS primarily causes disorders of histidine metabolism， tricarboxylic acid cycle， glyoxylate and dicarboxylate metabolism， as well as the imbalance of the activation/inactivation of endogenous metabolites， which may involve the immune response， material and energy metabolism， inflammatory response and intestinal flora， and may provide a basis for the establishment and application of SSDHS model.

Mycoplasma pneumoniae pneumonia complicated with top of the basilar syndrome in children: a case report / 临床儿科杂志

Objective To explore the clinical characteristics of Mycoplasma pneumoniae pneumonia (MPP) complicated with "top of the basilar" syndrome (TOBS) in children. Method The clinical data of MPP complicated with TOBS in a child were retrospectively analyzed, and the related literature was reviewed. Results A 6-year-old girl developed fever and inflexible movement in unilateral limb, and serum Mycoplasma pneumoniae antibody increased gradually. Lung CT showed a large area of inflammatory consolidation. Brain magnetic resonance imaging (MRI) showed multiple cerebral infarctions. After the symptomatic treatment of anti-infection, anticoagulation, thrombolysis and intracranial pressure reduction, the girl's condition continued to worsen and she presented with coma, cough weakness, dysphagia, facial nerve paralysis and bilateral pupillary inequality. Although the vital signs of the child were stable, she could not take care of herself and was still recovering. Conclusion MPP is often accompanied by hypercoagulative state and autoimmune abnormalities, and the prognosis of patients combined with TOBS is poor.

Effects of macrophage colony-stimulating factor on macrophage polarization, invasion and metastasis of ovarian cancer / 临床检验杂志

Objective@#To investigate the effects of macrophage colony-stimulating factor (M-CSF) on the polarization and infiltration of M2 macrophages and the invasion and metastasis of tumor cells in ovarian cancer microenvironment. @*Methods@#A co-culture system consisting of ovarian cancer cells (A2780 and SKOV3) and THP-1 derived macrophages was established in vitro. The M-CSF levels in culture medium and M-CSF mRNA levels in cancer cells and macrophages were detected by ELISA and qRT-PCR, respectively. The proportion of CD68+CD163+ M2 macrophages (polarization cells) was determined by flow cytometry. The invasive and metastatic ability of A2780 and SKOV3 cells after co-culturing with M2 macrophages were analyzed using Transwell assay. The expression levels of M-CSF, CD68+, CD163+ and E-cad in paraffin sections of 52 patients with ovarian cancer and 18 patients with benign ovarian tumor were detected by the immunohistochemistry staining, and their correlations and the relationship between M-CSF and clinicopathological features of ovarian cancer patients were analyzed. @*Results@#The M-CSF levels in culture medium of the co-culture group (A2780 and SKOV3 cells co-cultured with M2 macrophages) were significantly higher than that of A2780 and SKOV3 cells alone (t=14.315 and 12.338, P＜0.01). Fluorescence quantitative PCR results showed that the increased M-CSF originated from the secretion of co-cultured ovarian cancer cells (t=29.915 and 36.826, P＜0.01). The proportions of CD68+CD163+ M2 macrophages in the A2780 cells co-cultured with M2 macrophages group and SKOV3 cells co-cultured with M2 macrophages group were (6.14±0.50)% and (7.32±0.67)%, respectively, which were significantly higher than that in the M2 macrophages alone group ([1.82±0.34]%, t=12.289 and 12.711, P＜0.01). Transwell assay showed that the co-culture environment enhanced the invasion of A2780 and SKOV3 cells (24.00±4.81 vs 75.20±6.42, t=11.058; 18.40±2.31 vs 61.60±9.66, t=7.537, P＜0.01). The expression levels of M-CSF in ovarian cancer tissues were positively correlated with the number of CD68+ cells and CD163+ cells (r=0.690 and 0.596, P＜0.01), and negatively with the expression levels of E-cad (r=-0.566, P＜0.01). Moreover, the expression levels of M-CSF and the number of CD68+ cells and CD163+ cells in ovarian cancer tissues were significantly higher than that in benign ovarian tumor tissues, however, the expression levels of E-cad were on the contrary. The expression levels of M-CSF in ovarian cancer tissues were significantly correlated with tumor stage, differentiation and lymphatic node metastasis (χ2=6.240, 6.612 and 4.544, respectively, P＜0.05). @*Conclusion@#The increased expression of M-CSF in ovarian cancer microenvironment may induce the polarization and infiltration of CD68+CD163+ M2 macrophages, and then promote the invasion and metastasis of ovarian cancer cells.

National experts consensus on application of pulse contour cardiac output monitoring technique in severe burn treatment (2018 version) / 中华烧伤杂志

As a newly developed technique for hemodynamic monitoring, pulse contour cardiac output (PiCCO) monitoring takes great advantages in guiding shock resuscitation and fluid administration. PiCCO has been used more and more in burn patients in recent years, however there is no clinic consensus on how to apply PiCCO monitoring, understand the significance of PiCCO monitored parameters, and guide the treatment using PiCCO monitored parameters in patients with severe burns. Based on the current literature and the experts' clinical experience, (2018 ) is now issued by the Burn and Trauma Branch of Chinese Geriatrics Society, aiming to provide practical guidance for its usage in clinic.

Quantitative analysis of serum amino acid spectrum in osteoarthritis rats / 中华地方病学杂志

Objective To quantitatively analyze the dynamic changes of amino acid levels in serum of osteoarthritis rats,and to provide clues for further studies on biomarkers and metabolic mechanisms of osteoarthritis.Methods Sixty healthy male Sprague-Dawley (SD) rats were randomly divided into model group and control group according to their body weight by random number table method,the experimental period was 4,8 and 12 weeks,respectively,ten rats in each group.The left knee hind joints of rats in model group were operated by the modified Hulth method.After 5 days,rats of model group were driven to move for 30 minutes a day.Rats of two groups were fed ordinary solid fodder and drank tap water.At the end of the experimental period,the knee joints were collected for pathological observation.The amino acid content in serum was determined by high performance liquid chromatography-quadrupole ion trap tandem mass spectrometer (HPLC/Q-TRAP-MS/MS).The area under the receiver operating characteristic (ROC) curve (AUC) was used to determine the ability of differential amino acids to diagnose disease,AUC ＞ 0.7-0.9 was certain accuracy,＞ 0.9 was high accuracy.Results Under the light microscope,in control group,the articular cartilage surface was smooth and continuous,and the chondrocytes had good overall morphological structure and distinct layers;in model group,the articular cartilage of rats thinned,the surface was roughness and defect,the chondrocytes degenerated,necrosis and disappeared,and the lesions were aggravated with the extension of observation period.Forty kinds of amino acids were detected.Compared with control group,at 4 weeks after surgery,the level of 24 kinds of amino acids in model group decreased and 14 kinds of amino acids were increased,among which the differences in taurine,glutamine,serine,glutamic acid,aspartic acid,γ-aminobutyric acid and hydroxyproline were statistically significant (P ＜ 0.05);at 8 weeks after surgery,24 kinds of amino acids in model group were increased and 16 were decreased,among which the differences in histidine,serine,glutamic acid,homoarginine,lysine,isoleucine and glycine were statistically significant (P ＜ 0.05);at 12 weeks after surgery,36 kinds of amino acids in model group were decreased,among which the differences in 21 kinds of amino acids were statistically significant (P ＜ 0.05),all of their AUC were not less than 0.740,and the AUC of 14 amino acids were not less than 0.800,and the highest was kynurenine (0.980).Conclusions The model of knee osteoarthritis in rats is established successfully.With the increased time,the progress of osteoarthritis lesions increases,and the amino acid metabolism in osteoarthritis rats is out of order.

Screening of different metabolites in urine of children with Kaschin-Beck disease by the metabonomics technique / 中华地方病学杂志

Objective To search the different metabolites related with Kaschin-Beck disease (KBD) in urine of children with KBD by metabolomics technique,in order to provide clues in researching KBD biomarkers.Methods Among the children aged 7-15 years old in the KBD areas in Qinghai Province,the urine samples of 56 KBD children (case group),51 health children from the same area as the control group (internal control group) and 50 health children from non diseased areas (external control group) were examined by quadrupole-time of flight mass spectrometry (UPLC/QTOF-MS).Partial least squares discriminate analysis (PLS-DA) and multivariate statistical analysis were applied to explore and search the different endogenic metabolites associated with KBD.Results A obvious separation among three groups was observed,and the difference was significant among case group and internal,external control groups (P ＜ 0.05).Twenty-two different endogenic metabolites associated with KBD were found,compared with the external control group,21 metabolites were increased in the case group(P ＜ 0.05).Among them,14 kinds of metabolites (L-Cystine,isobutyryl carnitine,butyryl-L-carnitine,aspartyl-Asparagine,asparaginylAspartate and ubiquinone Q2,etc.) showed significant differences between the case group and internal control group (P ＜ 0.05).There were 4 kinds of metabolites showing significant differences between the two control groups(P ＜ 0.05).Condusion The metabolic profile in the urine of KBD children and healthy children is obviously different and the metabolites of L-Cystine,isobutyryl carnitine,butyryl-L-carnitine,aspartyl-Asparagine,asparaginyl-Aspartate and ubiquinone Q2 are more closely related with KBD,which can be considered as the clues in screening KBD biomarkers.

Screening of different metabolites in serum of children with Kaschin-Beck disease by the metabonomics technique / 中华地方病学杂志

Objective To search the different metabolites related with Kaschin-Beck disease (KBD) in serum of children with KBD. Methods Among the children aged 7 - 15 years old in the KBD areas in Qinghai Province,by the X ray and clinical examination,56 KBD patients(case group) and 51 non-KBD children (internal control group)were selected.Meantime,in non-KBD areas where the economic level and living habits was similar to that of the KBD areas, 50 healthy children were selected as external control group. Children's fasting blood samples were collected, and the metabolic profile in serum was determined by ultra performance liquid chromatography coupled with quadrupole-time of flight mass spectrometry (UPLC/QTOF-MS) technique. Combined with multivariate statistical analysis, database searching was applied to explore and search the different endogenic metabolites associated with KBD, to find biologically targeted makers. Results There were 1 384 serum metabolites with significant differences in case group, internal control group and external control group, 670 serum metabolites with significant differences in case group and external control group, including 25 KBD related endogenous differential metabolites. Among them, the relative contents of kynurenic acid and arachidonic acid in case group were higher than that of external control group, and the relative contents of N-α-acetylarginine, 6-hydroxymelatonin and sphinganine in case group were lower than that of external control group (P < 0.05). Conclusions The serum metabolic profile of children in KBD areas and children in non-KBD areas is significantly different. N-α-acetylarginine, kynurenic acid, 6-hydroxymelatonin, sphinganine and arachidonic acid are more closely related with KBD,and they can be considered as a clue in screening KBD biomarkers.

Effect of PIAS1 on Epithelial-mesenchymal Transition of Gastric Cancer under Inflammatory Microenvironment / 胃肠病学

Background:Protein inhibitor of activated STAT 1( PIAS1 )is an important regulator for inflammatory signaling network,which is low expressed in gastric cancer and associated with development of cancer,but its mechanism has not been elucidated. Aims:To investigate the effect of PIAS1 on epithelial-mesenchymal transition( EMT)of gastric cancer under inflammatory microenvironment. Methods:Recombinant adenovirus Ad5/F35-PIAS1 and Ad5/F35-null were constructed and transfected into gastric cancer cell line SGC-7901,mRNA and protein expressions of PIAS1 were detected by RT-PCR and Western blotting,respectively. SGC-7901 cells were divided into IL-6 treatment group,Ad5/F35-PIAS1﹢IL-6 treatment group and Ad5/F35-null﹢IL-6 treatment group. Cell proliferation was measured by MTT method,migration and invasion capacities were assessed by wound healing test and Transwell chamber invasion assay. Protein expressions of E-cadherin,Snail,Twist,Vimentin and P-p38MAPK were assessed by Western blotting. Results:The transfection of Ad5/F35-PIAS1 significantly increased the expressions of PIAS1 mRNA and protein in SGC-7901 cells. Compared with IL-6 treatment group and Ad5/F35-null﹢IL-6 treatment group,capacities of cell proliferation,migration and invasion were significantly decreased(P ﹤0. 01);protein expressions of Snail,Twist,Vimentin and P-p38MAPK were significantly decreased while expression of E-cadherin protein was significantly increased in Ad5/F35-PIAS1 ﹢IL-6 treatment group ( P﹤0. 01). No significant differences in above-mentioned indices were found between IL-6 treatment group and Ad5/F35-null﹢IL-6 treatment group(P﹥0. 05). Conclusions:PIAS1 could inhibit EMT of gastric cancer cells under inflammatory microenvironment,and may play an important role in inhibition of tumor invasion and metastasis.

Urinary metabolic profiling of rats with articular cartilage damage induced by T-2 toxin / 中华地方病学杂志

Objective To search for the metabolites that associated with articular cartilage damage in the urine of rat model with articular cartilage destruction induced by T-2 toxin.Methods Thirty healthy male Wistar rats aged 4-5 weeks were numbered by weight,randomly divided into two groups (n =15 per group),namely the control group and the model group.The rats in the control group were fed with standard rat diets,and those in the model group were given diets contaminated by T-2-toxin (300 μg/kg).Throughout the experiment,all animals were given free access to distilled water and diets.After continuous treatment for 3 months,all the rats were sacrificed.The changes of articular cartilage in rat knee joints were observed by histopathological method,the metabolic profile of rats' urine was determined by ultra performance liquid chromatography/quadrupole time of flight-mass spectrometry (UPLC/QTOF-MS) technique.Combined with multivariate statistical analysis,database searching was applied to explore and confirm the different metabolites associated with cartilage damage.Results Light microscope showed that rats' articular chondrocytes in the control group presented cells in neat rows and eumorphism,rats' articular chondrocytes in the model group presented extensive areas of chondrocyte degeneration,necrosis and loss.In rats' urine metabolic profiles,5 different metabolites associated with cartilage destruction were detected,such as 4-hydroxynonenal (HNE),trans-4,5-epoxy-2(E)-decenal (EDE),5-methyldeoxycytidine,and 5-L-glutamyl-glycine and prolyl-valine.Compared with the control group (mass spectrum peak area:65820 ± 5200,22080 ± 3538,4292 ± 3520,3277 ± 2025,1104 ± 990),all of them increased in the model group (mass speetrum peak area:90240 ± 18863,25610 ± 5071,9702 ± 6562,6029 ± 3905,4144 ± 5322,t =-3.903,-2.209,-2.814,-2.424,-2.174,all P ＜ 0.05).Conclusions The articular cartilage destruction induced by T-2 toxin could cause the changes of related metabolites in the urine;the 5 kinds of changed metabolites in urine are related to articular cartilage destruction.

A comparative study between minimally invasive and traditional open surgery in treating patients with pancreatic body or tail lesions / 中华肝胆外科杂志

Objective To study the feasibility,safety,indications and possible advantages of minimally invasive surgery over traditional open surgery in treating pancreatic body or tail lesions.Methods From December 2009 to December 2014,the clinical data of 71 patients with lesions in pancreatic body or tail who underwent minimally invasive distal pancreatectomy (MIDP) or open distal pancreatectomy (ODP) at the General Surgery of Huadong Hospital were retrospectively analyzed.There were 22 patients in the MIDP group and 49 patients in the ODP group.The operations in 15 patients in the MIDP group were performed by the Da Vinci robot-assisted surgical system and 7 patients by laparoscopic distal pancreatectomy.Results The MIDP group had a shorter time to pass first flatus [(MIDP (2.5 ± 1.0) d vs ODP (3.5 ±1.0)d,P ＜ 0.05],and shorter postoperative hospital stay [(MIDP (15.2 ± 7.9) d vs ODP (23.4 ±21.2) d,P ＜ 0.05] than the ODP group.There were no significant differences on total pancreatic fistula rate [MIDP 45.5％ (10/22) vs ODP 55.1％ (27/49),P ＞ 0.05] and symptomatic postoperative pancreatic fistula rate [MIDP 18.2％ (4/22) vs ODP 18.4％ (9/49),P ＞ 0.05] between the two groups.The MIDP group had a significant longer operative time [MIDP (246.3 ±75.3)min vs ODP (168.1 ±33.7)min,P＜0.05] than the ODP group.Conclusions Minimally invasive surgery is safe and feasible in treatment of lesions in pancreatic body or tail with less trauma and faster recovery.The application of robotic surgery has expanded the treatment options for lesions in pancreatic body or tail.

Analysis of the clinical diagnosis and therapy and misdiagnosis for congenital middle ear cholesteatoma / 中国耳鼻咽喉头颈外科

[ABSTRACT]OBJECTIVETo investigate the methods of diagnosing and treating congenital middle ear cholesteatoma and causes of misdiagnosis.METHODSThis was a retrospective review of 9 cases of congenital middle ear cholesteatoma in this hospital.RESULTS9 patients all received operative treatment and definite diagnosis was made by histopathologic investigation. The ossicular reconstruction was performed to 6 patients. All of the cases were followed-up for 3 months-2.5 years. A-B gap was 20 dB. After 1 year, no residual or recurrence of cholesteatoma was found in CT scan. All patients had been misdiagnosed. Misdiagnosis rate was 100%.CONCLUSIONCongenital middle ear cholesteatoma is a clinically rare and diagnosis is usually delayed in clinical practice due to the silent nature of the disease in its early stage. Preoperative diagnosis may be based on Levenson diagnostic criteria and CT imaging. Early surgical treatment can obtain a good effect of hearing reconstruction. To avoid misdiagnosis and incorrect treatment, radiological evaluation and careful local examination are really important, which may also avoid serious complications.

Significance of S-100 and CD83+DC Expression in Laryngeal Mucosa Lesions / 天津医药

Objective To investigate the infiltration of dendritic cell (DC) in Vocal cord polyp, laryngeal leukopla-kia and glottic squamous cell carcinoma,and to observe laryngeal mucosal lesions of the state of the immune microenviron-ment, and to research significance of DC on the development of glottic squamous cell carcinoma. Methods The infiltration of S-100+and CD83+DC in 20 cases of Vocal cord polyp, 47 cases of laryngeal leukoplakia, 45 cases of glottic squamous cell carcinoma tumor and 20 cases of laryngeal normal mucosa were examined using immunohistochemistry. Results The num-ber of S-100+and CD83+DC were significantly higher in glottic squamous cell carcinoma, laryngeal leukoplakia and vocal cord polyp than that in laryngeal normal mucosa (P<0.05). The number of S-100+and CD83+DC were higher in laryngeal leukoplakia than that in glottic squamous cell carcinoma (P<0.05). The number of S-100+and CD83+DC in mild dysplasia and moderate dysplasia were less than that in severe atypical hyperplasia (P<0.01). In glottic squamous cell carcinoma, S-100+ and CD83+DC with poor-differentiated was significantly less than that with well-differentiated (P < 0.01). Conclu-sion Changes in the number of dendritic cell was found in vocal cord polyp, laryngeal leukoplakia and glottic squamous cell carcinoma, which indicated that there were an abnormal immune status. Changing of dendritic cell in laryngeal mucosa plays an important role in laryngeal cancer development.

The relationship between vascular endothelial growth factor C and recurrence following curative resection of pancreatic cancer / 中华肝胆外科杂志

Objective To investigate the prognostic value of vascular endothelial growth factor C (VEGF-C) and clinicopathologic indexes in predicting recurrence following curative resection of pancreatic cancer. Methods The expressions of VEGF-C of 47 patients who underwent curative resection for curative pancreatic cancer resection were detected by Envision immunohistochemical methods. The effects of VEGF-C and clinicopathologic indexes on recurrence were assessed by the Kaplan-Meier and Cox proportional hazards model. Results The positive rates of VEGF-C were 61. 7% in = 29) and 14. 9%(n = 7), respectively, in pancreatic cancer and normal pancreatic tissues. The positive expression of VEGF-C in pancreatic carcinoma was obviously higher than the normal pancreatic tissues (P = 0. 018). The median disease-free survival time was 11. 9 months, the average disease-free survival time was 18. 4 + 2. 4 months, and the cumulative 1-year, 2-year and 3-year actuarial recurrence free survival rates were 46. 8%, 23. 4%, 14. 4%, respectively. There was a significant correlation between the VEGF-C expression and lymph node metastasis in pancreatic cancer (P = 0. 036). On Kaplan-Meier analysis, VEGF-C (P = 0. 020), tumor diameter (P = 0. 013), age (P = 0. 057) and adjuvant chemotherapy (P=0. 017) were associated with disease-free survival time. Multivariate analysis showed VEGF-C (P = 0. 009), tumor diameter (P = 0. 010) and adjuvant chemotherapy (P = 0. 017)were independent prognostic factors of disease-free survival after surgery for pancreatic cancer.Conclusion The expression of VEGF-C was higher in pancreatic cancer, and VEGF-C was correlated with lymph node metastasis. VEGF-C was the biomarker that independently predicted disease-free survival after surgery for pancreatic cancer.

Measurement of the expression level of guanylyl cyclase-C in the peripheral blood mononuclear cell by real-time fluorenscence quantitative method / 临床检验杂志

Objectives To establish real-time fluorenscence quantitative polymerase chain reaction ( RFQ-PCR ) for measurement of the expression level of guanylyl cyclase-C(GC-C) in the Peripheral blood mononuclear cell(PBMC)in 30 blood donors, 10 cases colorectal cancer tissue and 1 case T84 human colon cancer cell line. Methods Specific primers and TaqMan probe have been designed,and fluorenscence of the PCR product was detected continuously during amplification. According to the standard curve created by plasmid DNA, the expression level of GC-C in clinical samples has been determined using software, and the results were presented as the ratios of GC-C mRNA to?2-microgluobulin(?2M)mRNA. Results The detection range of the assay was from 101 pg/ml to109pg/ml,the coefficient of variation values of both intra-experimental and inter- experimental reproducibility were 6. 87% to 11. 12% and 8. 86% to 15. 19% . None of 30 blood donors and 11 benign intestinal patients expressed GC-C mRNA,it was expressed in 31/37 colorectal cancer patients. The expression level of GC-C mRNA in colorectal cancer patients was 0. 88?0.06,and the expression level of its in colorectal carcinoma tissue and T84 cells were 0. 86?0.07/ug tissue and 0.0082/per cell. Conclusions This assay had high sensitivity,specificity and reproducibility.