Comparison of performance for leukocyte differential counting of the Technicon H6000 system with a manual reference method using the NCCLS standard

The National Committee for Clinical Laboratory Standards (NCCLS) has published a tentative standard for leukocyte differential counting, by means of which a manual or automated method for leukocyte differential counting can be compared with a manual reference method. The performance of the Technicon H6000 system was evaluated using the standard at Stamford and Overlook Hospitals. A total of 502 patient samples were analyzed: 315 from Overlook and 187 from Stamford. The H6000 system was found to be approximately four times more precise than the 200-cell manual reference method for each cell type. Correlation of the H6000 system with the manual method was good, with correlation coefficients of 0.98 for neutrophils and lymphocytes, 0.96 for eosinophils, 0.72 for monocytes, and 0.5 for basophils. The clinical sensitivity of the H6000 system, measured in terms of false normals and false abnormals, was similar to that of the manual reference method when measured against itself. There were no clinically significant discrepancies in results from the H6000 system, except for possibly one case where a patient was already on antibiotic therapy. The NCCLS standard was found to be a useful but rather complex and involved method for evaluating the performance of the H6000 system, the major problem being the amount of work needed to count manually the number of cells required for the manual reference method.     

Multicenter evaluation of commercial frozen plates for microdilution broth antifungal susceptibility testing of yeasts and comparison of MIC limits recommended in NCCLS M27-A2

A commercial kit, Frozen Plate for Antifungal Susceptibility Testing of Yeasts, Eiken (Eiken Chemical Co., Ltd., Tokyo), was tested in a multi-institute study to evaluate the agreement between interinstitute MICs and National Committee for Clinical Laboratory Standards (NCCLS) M27-A2 recommendation limits of MIC value. The kit was reported as a method equivalent to the standardized guidelines for antifungal susceptibility testing by the Committee for Clinical Laboratory Standards-1994, the Japanese Society for Medical Mycology, and which is widely used in Japan for amphotericin B, flucytosine, fluconazole, miconazole, and itraconazole. The degrees of inter-institute and NCCLS agreements were good to excellent especially with 48-hr incubation for all antifungal agents. However, the percent agreements to NCCLS recommendations against itraconazole were poor. Overall, MIC values obtained using the frozen plate antifungal susceptibility testing kit, with 48-hr incubation, were thought to be reliable and convenient alternatives to the data obtained by the NCCLS M27-A2 reference macrodilution and microdilution method. This kit will allow matching of results between international laboratories. However, the MIC value for itraconazole requires careful interpretation.     

Use of pattern recognition technology for determination of the human differential leukocyte count

The imprecision, inaccuracy, and clinical sensitivity of an automated differential counter based on pattern recognition technology (HEMATRAK model 590) were evaluated using the NCCLS standard H20-T. The data show that the instrument is capable of performing assays in a manner comparable with the reference method proposed by NCCLS. In addition, data are presented from a multicenter study involving 174 HEMATRAK instruments which showed that the automated systems produce results equivalent to manual procedures (based on approximately 350 individual assays).     

Establishment and identification of the human multi-drug-resistant cholangiocarcinoma cell line QBC939/ADM

In this study, we aim to establish the human multi-drug-resistant cholangiocarcinoma cell line QBC939/ADM which can be grow and passaged steadily in 1 μg/ml concentration of adriamycin in appropriate medium. The human multi-drug-resistant cholangiocarcinoma cell line QBC939/ADM was established using the method of exposure to medium with adriamycin alternated between high and low concentration with gradually increasing concentration. Furthermore, QBC939 and QBC939/ADM were both treated with adriamycin, mitomycin and vindesine, and then detected by MTT assay, respectively. Growth cycle and intra-cellular concentrations of ADM within cells of each group were determined by flow cytometry. Expression levels of P-glycoprotein were detected by Western bolt and real-time PCR. Results showed that, compared with QBC939, the inhibitive rates of adriamycin, mitomycin and vindesine to QBC939/ADM were lower. Content of ADM in the QBC939/ADM was lower. Western bolt and real time PCR showed that P-glycoprotein in the QBC939/ADM group was over expressed. Therefore, QBC939/ADM was establish and identified as the multi-drug-resistant cell line, which can grow and be passaged steadily in 1 μg/ml concentration adriamycin in appropriate medium. And the multi-drug-resistant character of QBC939/ADM was indicated to be related to the over expression of P-glycoprotein induced by chemotherapeutic drugs.     

Modification of the fluorescein diacetate assay for screening of antifungal agents against Candida albicans: comparison with the NCCLS methods

A modified fluorescein diacetate (FDA) assay has been compared with standard NCCLS broth macrodilution and broth microdilution methods for the detection of antifungal activity. The FDA assay was performed in a medium containing bacteriological peptone, NaCl, yeast extract and glucose (0.2%, 0.1%, 0.1% and 1% w/v, respectively) and buffered with 10 mM BES buffer. The MICs of amphotericin B, fluconazole, miconazole and flucytosine (representing three major classes of antifungal agents) obtained by the three methods were compared. The results obtained with the FDA assays correlated well with the NCCLS macrodilution method for MICs of amphotericin B, miconazole and fluconazole, but not for flucytosine. However, the MIC values of flucytosine obtained with the FDA assay were well within the quality control range for the two reference strains recommended by the NCCLS. The FDA assay described is an attractive alternative to the NCCLS methods for screening for antifungal agents, with the added advantage of objectivity of fluorescence measurement.     

Rationale for the NCCLS standard H20-T for differential leukocyte counting

The National Committee for Clinical Laboratory Standards (NCCLS) tentative standard for Leukocyte Differential Counting (H20-T) was developed in the United States as a reference method for differential leukocyte counting as well as a method for the evaluation of the performance of differential leukocyte counters. The key features of this document are outlined and the reasons for their inclusion in the standard are given.     

铜绿假单胞菌的院内感染分布及耐药性的调查

目的了解铜绿假单胞菌（PA）在医院内感染的分布特点及其耐药性,指导临床合理用药,降低医院感染率。方法回顾分析2005年1月至2008年11月我院临床分离的铜绿假单胞菌152株,按WHO推荐的NC-CLS标准方法（K—B法）进行药敏试验,分析菌株的临床分布特征及体外药敏结果。结果铜绿假单胞菌在临床上主要以呼吸道感染为主。在17种临床常用抗生素中,多表现为多重耐药。美洛培南、亚胺培南及左旋氧氟沙星抗PA效果最好,耐药率分别只有14．47％、14．47和5．26％。结论多重耐药铜绿假单胞菌在临床广泛存在,应当根据临床体外药敏结果合理选择抗菌药物进行治疗,以取得良好临床效果并减少耐药菌产生;同时规范院内感染控制措施,减少耐药菌株的播散。     

Use of spectrophotometric reading for in vitro antifungal susceptibility testing of Aspergillus spp

A comparative study of visual and spectrophotometric MIC endpoint determinations for antifungal susceptibility testing of Aspergillus species was performed. A broth microdilution method adapted from the National Committee for Clinical Laboratory Standards (NCCLS) was used for susceptibility testing of 180 clinical isolates of Aspergillus species against amphotericin B and itraconazole. MICs were determined visually and spectrophotometrically at 490 nm after 24, 48, and 72 h of incubation, and MIC pairs were compared. The agreement between the two methods was 99% for amphotericin B and ranged from 95 to 98% for itraconazole. It is concluded that spectrophotometric MIC endpoint determination is a valuable alternative to the visual reference method for susceptibility testing of Aspergillus species.     

Detection of two-component mixtures of lognormal distributions in grouped, doubly truncated data: analysis of red blood cell volume distributions

We have examined the statistical requirements for the detection of mixtures of two lognormal distributions in doubly truncated data when the sample size is large. The expectation-maximization algorithm was used for parameter estimation. A bootstrap approach was used to test for a mixture of distributions using the likelihood ratio statistic. Analysis of computer simulated mixtures showed that as the ratio of the difference between the means to the minimum standard deviation increases, the power for detection also increases and the accuracy of parameter estimates improves. These procedures were used to examine the distribution of red blood cell volume in blood samples. Each distribution was doubly truncated to eliminate artifactual frequency counts and tested for best fit to a single lognormal distribution or a mixture of two lognormal distributions. A single population was found in samples obtained from 60 healthy individuals. Two subpopulations of cells were detected in 25 of 27 mixtures of blood prepared in vitro. Analyses of mixtures of blood from 40 patients treated for iron-deficiency anemia showed that subpopulations could be detected in all by 6 weeks after onset of treatment. To determine if two-component mixtures could be detected, distributions were examined from untransfused patients with refractory anemia. In two patients with inherited sideroblastic anemia a mixture of microcytic and normocytic cells was found, while in the third patient a single population of microcytic cells was identified. In two family members previously identified as carriers of inherited sideroblastic anemia, mixtures of microcytic and normocytic subpopulations were found. Twenty-five patients with acquired myelodysplastic anemia were examined. A good fit to a mixture of subpopulations containing abnormal microcytic or macrocytic cells was found in two. We have demonstrated that with large sample sizes, mixtures of distributions can be detected even when distributions appear to be unimodal. These statistical techniques provide a means to characterize and quantify alterations in erythrocyte subpopulations in anemia but could also be applied to any set of grouped, doubly truncated data to test for the presence of a mixture of two lognormal distributions.     

IL-6 对胆管癌细胞凋亡的作用机制研究

目的：探讨不同浓度白细胞介素6（IL-6）对胆管癌细胞的凋亡作用及其机制。方法：采用胆管癌细胞株QBC939 进行试验, 用MTT 法检测不同浓度IL-6 对人胆管癌细胞系QBC939 增殖的影响,用Annexin V和PI染色检测不同浓度IL-6对人胆管癌细 胞系QBC939 凋亡的影响,RT-PCR 检测细胞内Bcl-2、Bax 的mRNA水平,并且将QBC939 细胞移植至裸鼠皮下观察移植瘤的 生长速度、重量以及体积的变化。结果：同时间段不同浓度IL-6 的QBC939 细胞增殖明显高于对照组（P<0.05）,且IL-6 的浓度越 高,其增殖的水平也越高,OD 值与IL-6 的浓度呈正相关关系;不同浓度IL-6 对胆管癌细胞的凋亡率较对照组明显下降 （P<0.05）,且随着IL-6 的浓度不断的增高,细胞的凋亡率逐渐下降,IL-6 的浓度与细胞的凋亡率呈负相关关系;对照组细胞培养 48 h 后的凋亡率显著高于培养24 h后（P<0.05）,而IL-6 实验组的细胞的24 h凋亡率与48 h凋亡率差异无统计学意义（P>0.05）。 Bcl-2 的mRNA水平随着IL-6 浓度增加逐渐升高,而Bax 的mRNA水平随着IL-6 的浓度增加逐渐降低,且IL-6 浓度与Bcl-2 mRNA水平呈正相关关系,IL-6 的浓度与Bax mRNA水平呈负相关关系。IL-6 实验组的肿瘤体积和重量与对照组相比均明显增 大（P<0.05）,且IL-6 实验组移植瘤的生长速度明显高于对照组（P<0.05）。结论：IL-6 能够促进QBC939细胞增殖,抑制其凋亡,可 能与上调Bcl-2 的mRNA水平以及下调Bax的mRNA水平有关。     

Occurrence of beta-lactamase type ESBL and IBL in Pseudomonas aeruginosa rods]

The aim of the study was to determine extended spectrum beta-lactamases (ESBL) and inducible beta-lactamases (IBL) among Pseudomonas aeruginosa strains. A total of 43 strains isolated from humans (6), hospital sink (1), fish (15), cattle (5), swine (5), dog (1), redder (1) fur animals (9) were studied. ESBL-producing strains were detected with double disc diffusion test according to Jarlier et al. (8). Clavulonate and tazobactam were used as the inhibitors of ESBL. Inducible beta-lactamases were determined using double disc method according to Sanders (15). Cefoxitin was the inductor of these beta-lactamases. The susceptibility study was carried out using the disc diffusion method according to NCCLS standards. A total of 8 ESBL (18.6% of all strains) and 31 (72%) IBL producing strains were detected. The obtained results indicate the necessity of monitoring of ESBL- and IBL-producing strains of Pseudomonas aeruginosa.     

A comparison of thick smears, QBC malaria, PCR and PATH falciparum malaria test trip in Plasmodium falciparum diagnosis.

Blood samples from 182 patients presenting at the out-patient clinic in Richard-Toll. Senegal were analysed by Thick smear microscopy, the QBC, PCR and the new dipstick PATH Malaria assay which detects the histidine rich protein II antigen of Plasmodium falciparum. Thick smear microscopy was used as the reference method. Sensitivity, specificity, predictive positive and negative values were 100%, 83.6%, 93.4% and 100% QBC respectively; 100%, 72.7%, 89.4% and 100% for PCR; 96%, 92.7%, 96.8% and 91% for the PATH assay. PATH assay failed to detect one positive sample with Plasmodium malariae. Assays were also compared with regard to the expense of equipment and reagents and speed and ease of use. The rapid PATH assay can be performed with minimal training and may be specially useful in areas where P. falciparum is the predominant malaria species, in epidemic malaria regions, and where skilled microscopy is not readily available.     

Minimal inhibitory concentrations of Candida species to five antifungals by using the reference dilution micromethod and the E-test.

It is accepted that the frequency of candidosis has increased during the last decade, specially in hospitalized patients. The more frequent use of azole antifungals and the recognition of isolates of Candida sp resistant to these and other drugs such as 5-fluorocytosine constitute a great need for a reproducible and useful C. albicans in vitro susceptibility testing method for monitoring antifungal therapy in clinical mycological laboratories. The E-test is a novel agar diffussion technique for testing the susceptibility of yeasts against a defined continous gradient of drug and could be used by most clinical laboratories. In this study the E-test and the NCCLS reference microbroth method (M27-P guidelines) were used to determine the MICs of amphotericin B, 5-flucytosine, itraconazole, fluconazole and ketoconazole for 50 clinical isolates of Candida albicans, Torulopsis glabrata, C. tropicalis and Hansenula anomala and five reference ATCC strains. The main purpose of the study was to compare the results obtained by the two methods. In general good agreement (+/- 1 dilution) was otained between both methods, despite differences observed for some species-antifungal combinations in which the MICs were lower by the E-test than by the microbroth method. MICs for C. albicans and T. glabrata to amphotericin B were < 0.50 microg/mL. Two isolates of C. albicans and two others of H. anomala, showed MIC < 8 microg/mL for 5- flucytosine. All isolates of T. glabrata and 40% of C. albicans showed MICs > 16 microg/mL for fluconazole. The results of this study indicate that E-test is an alternative for susceptibility testing to the NCCLS reference method. Because its simplicity it seems to be an easier test for routine clinical laboratories.     

Down-regulation of c-Myc expression inhibits the invasion of bile duct carcinoma cells

Cholangiocarcinoma is the second most common primary hepatic tumour originating from biliary tract epithelial cells with poor prognosis. Enhanced c-Myc protein expression contributes to many aspects of tumour cell biology. Although the ability of c-Myc to drive unrestricted cell proliferation and to inhibit cell differentiation had been well recognized, whether down-regulated c-Myc expression can inhibit tumour cell invasion still remains to be explored. The c-Myc ASODN (antisense oligodeoxyribonucleotide) and NSODN (nonsense oligodeoxyribonucleotide) were designed, synthesized and transfected into human QBC939 bile duct carcinoma cells using the Lipofectamine 2000 reagent. The protein expression of c-Myc was detected by Western blot. A transwell experiment was applied to evaluate the invasive capacity of the QBC939 cells. c-Myc ASODN could significantly suppress the c-Myc protein expression (P<0.05) and the invasion (P<0.01) of QBC939 cells transfected with c-Myc ASODN compared with that in the control and c-Myc NSODN-transfected group. Thus in the present study we show that down-regulation of c-Myc expression can inhibit the invasion of QBC939 cells in vitro.     

Application of digital image analysis and flow cytometry to enumerate marine viruses stained with SYBR gold

A novel nucleic acid stain, SYBR Gold, was used to stain marine viral particles in various types of samples. Viral particles stained with SYBR Gold yielded bright and stable fluorescent signals that could be detected by a cooled charge-coupled device camera or by flow cytometry. The fluorescent signal strength of SYBR Gold-stained viruses was about twice that of SYBR Green I-stained viruses. Digital images of SYBR Gold-stained viral particles were processed to enumerate the concentration of viral particles by using digital image analysis software. Estimates of viral concentration based on digitized images were 1.3 times higher than those based on direct counting by epifluorescence microscopy. Direct epifluorescence counts of SYBR Gold-stained viral particles were in turn about 1.34 times higher than those estimated by the transmission electron microscope method. Bacteriophage lysates stained with SYBR Gold formed a distinct population in flow cytometric signatures. Flow cytometric analysis revealed at least four viral subpopulations for a Lake Erie sample and two subpopulations for a Georgia coastal sample. Flow cytometry-based viral counts for various types of samples averaged 1.1 times higher than direct epifluorescence microscopic counts. The potential application of digital image analysis and flow cytometry for rapid and accurate measurement of viral abundance in aquatic environments is discussed.     

Application of Digital Image Analysis and Flow Cytometry To Enumerate Marine Viruses Stained with SYBR Gold

A novel nucleic acid stain, SYBR Gold, was used to stain marine viral particles in various types of samples. Viral particles stained with SYBR Gold yielded bright and stable fluorescent signals that could be detected by a cooled charge-coupled device camera or by flow cytometry. The fluorescent signal strength of SYBR Gold-stained viruses was about twice that of SYBR Green I-stained viruses. Digital images of SYBR Gold-stained viral particles were processed to enumerate the concentration of viral particles by using digital image analysis software. Estimates of viral concentration based on digitized images were 1.3 times higher than those based on direct counting by epifluorescence microscopy. Direct epifluorescence counts of SYBR Gold-stained viral particles were in turn about 1.34 times higher than those estimated by the transmission electron microscope method. Bacteriophage lysates stained with SYBR Gold formed a distinct population in flow cytometric signatures. Flow cytometric analysis revealed at least four viral subpopulations for a Lake Erie sample and two subpopulations for a Georgia coastal sample. Flow cytometry-based viral counts for various types of samples averaged 1.1 times higher than direct epifluorescence microscopic counts. The potential application of digital image analysis and flow cytometry for rapid and accurate measurement of viral abundance in aquatic environments is discussed.     

A study of the association between schizophrenia and the dopamine D3 receptor gene

A study of the genetic association between schizophrenia and aBalI polymorphism in exon 1 of the dopamine D₃ (DRD3) gene, a candidate gene for schizophrenia, was conducted. The polymorphism was examined in 91 patients whose symptoms satisfied DSM-III-R for schizophrenia and 90 controls. There were no significant differences between the groups in allele frequencies or genotype counts. Contrary to a previous report, the patients were no more likely to be homozygous than controls. Moreover, no association with the presence of illness could be demonstrated when the patients were grouped according to sex, age of onset, history of admission to psychiatric institutions or positive family history.     

Lack of correlation between seminal and plasma HIV-1 viral loads is associated with CD4 T cell depletion in therapy-naïve HIV-1+ patients

The present study was conducted to investigate a possible correlation between plasma (PVL) and seminal viral load (SVL) on treatment-na?ve HIV-1-infected patients in Vitória, ES, Brazil. We also evaluated whether the progressive immunosuppression associated with HIV disease (as evidenced by declining CD4 T cell counts) has any impact on the correlation between PVL and SVL HIV-1. Viral load on paired blood and semen samples from 56 consecutive treatment-na?ve patients were evaluated and compared to CD4 cell counts. Viral load and T cell counts (cells/microl) were determined by NASBA and by flow cytometry, respectively. Overall, a strong positive correlation between PVL and SVL (rho = 0.438, p = 0.001) was observed. However, when patients were grouped according to their CD4 counts, this correlation was only significant among patients with CD4 counts > 200 cells/microl. Results presented here demonstrate the existence of a strong correlation between PVL and SVL on patients with CD4 cell counts > 200 cells/microl, suggesting that this association may correlate with disease progression.     

Analysis of the volume of red blood cells: application of the expectation-maximization algorithm to grouped data from the doubly-truncated lognormal distribution

In accordance with general principles recommended by the International Committee for Standardization in Haematology (1982, Journal of Clinical Pathology 35, 1320-1322), we have developed statistical methods for the analysis of red cell volume distributions. To select an appropriate reference distribution for goodness-of-fit testing, we derived a mathematical model of erythropoiesis that predicted a lognormal form for the distribution of erythrocyte volumes. Model predictions were then tested using samples obtained from 50 healthy individuals. Each grouped red cell volume distribution was doubly-truncated to eliminate artifactual frequency counts. Distribution parameter estimates were computed using the expectation-maximization algorithm, a missing information technique. Results of the one-sample chi-square goodness-of-fit test showed a fairly even distribution of P-values over the interval. Examples of the application of these statistical procedures to distributions from patients with anemia are given. Our results suggest that, for the analysis of red blood cell volumes, (i) parameter estimation should be made with the expectation-maximization method, and (ii) the truncated lognormal distribution should be used as a reference distribution for goodness-of-fit testing. This method could be applied to any set of grouped doubly-truncated data which, after transformation, follows the normal model.     

On-site diagnosis of Plasmodium falciparum, P. vivax, and P. malariae by using the Quantitative Buffy Coat system.

The Quantitative Buffy Coat (QBC) system was used for the detection and identification of malaria parasites in blood specimens from 570 residents of Oksibil, an isolated highland valley in the eastern Jayawijaya Mountains of Irian Jaya (Indonesian New Guinea). The availability of a battery-powered centrifuge and a fiberoptic Paralens enabled us to complete and interpret the assay in this remote environment. Of 322 QBC tubes examined for 2-4 min each, results of 295 (92%) concurred with findings on the matched Giemsa-stained thick smear (GTS). The 27 discrepant results included 13 QBC+/GTS- that, upon reexamination, were found to be GTS+. When using the corrected GTS results as the standard, the sensitivity and specificity of the QBC were 94% and 96%, respectively. Because electricity was available only 3 hr per day, it was decided to decrease the examination for an additional 248 QBC to a maximum of 90 sec per tube. This shortened inspection time resulted in a reduction of sensitivity to 53% but specificity was preserved at 89%. Forty-two of 45 conflicting results, QBC-/GTS+ from cases of light Plasmodium falciparum infections with < 1 trophozoite or gametocyte per field, were resolved by reexamination of the QBC in the laboratory. Tubes held at 4 C could be reexamined, without noticeable loss of fluorescence, for at least 6 wk after collection. Despite some difficulty in the identification of Plasmodium species, it was concluded that the QBC is an easy, sensitive method for the rapid diagnosis of malaria in the field and that it provides the inexperienced microscopist with an additional means for on-site identification of individuals needing treatment.