Assessment of aldehyde dehydrogenase in viable cells

Cytosolic aldehyde dehydrogenase (ALDH), an enzyme responsible for oxidizing intracellular aldehydes, has an important role in ethanol, vitamin A, and cyclophosphamide metabolism. High expression of this enzyme in primitive stem cells from multiple tissues, including bone marrow and intestine, appears to be an important mechanism by which these cells are resistant to cyclophosphamide. However, although hematopoietic stem cells (HSC) express high levels of cytosolic ALDH, isolating viable HSC by their ALDH expression has not been possible because ALDH is an intracellular protein. We found that a fluorescent aldehyde, dansyl aminoacetaldehyde (DAAA), could be used in flow cytometry experiments to isolate viable mouse and human cells based on their ALDH content. The level of dansyl fluorescence exhibited by cells after incubation with DAAA paralleled cytosolic ALDH levels determined by Western blotting and the sensitivity of the cells to cyclophosphamide. Moreover, DAAA appeared to be a more sensitive means of assessing cytosolic ALDH levels than Western blotting. Bone marrow progenitors treated with DAAA proliferated normally. Furthermore, marrow cells expressing high levels of dansyl fluorescence after incubation with DAAA were enriched for hematopoietic progenitors. The ability to isolate viable cells that express high levels of cytosolic ALDH could be an important component of methodology for identifying and purifying HSC and for studying cyclophosphamide-resistant tumor cell populations.     

Accuracy of supplementary serologic testing for human T-lymphotropic virus types I and II in US blood donors. Retrovirus Epidemiology Donor Study

Blood donations in the United States have been screened for antibody to human T-lymphotropic virus type I (HTLV-I) by HTLV-I enzyme immunoassay (EIA) since November 1988. Specimens repeatedly found to be reactive by EIA undergo confirmation by supplementary serologic tests. We assessed the accuracy of blood center testing of 994 HTLV-I EIA repeat-reactive specimens in five US blood centers between November 1988 and December 1991. Of 410 confirmed HTLV-I/II donations, 407 (99.3%) were infected with HTLV-I/II, as determined by polymerase chain reaction (PCR) (403 cases) and by repeat serologic testing (4 cases). The three false- positive results occurred in the first year of testing. Of 425 HTLV- indeterminate specimens, 6 (1.4%) were found to be infected by PCR (5 with HTLV-II and 1 with HTLV-I). None of 159 confirmatory test-negative donations was PCR positive. Of HTLV-I/II-seropositive specimens, 80.2% to 95.4% could be typed as HTLV-I or HTLV-II by type-specific serologic assays. These results support recommendations that HTLV-I/II- seropositive donors should be advised that they are infected with HTLV- I, HTLV-II, or HTLV-I/II (depending on results of type-specific assays). HTLV-indeterminate donors should be advised that their results only rarely indicate HTLV infection. HTLV confirmatory test-negative donors should be reassured that they are not infected with HTLV-I or HTLV-II.     

股骨近端锁定加压钢板治疗股骨转子间骨折

目的 探讨股骨近端锁定加压钢板（LCP）内固定治疗股骨转子间骨折的临床疗效.方法 采用股骨近端LCP治疗23例股骨转子间骨折的患者.随访观察骨折愈合时间,按Harris评分标准评价疗效.结果 23例均获随访,时间6~12（9±1.4）个月.骨折愈合时间14~20（17±1.7）周.髋关节Harris评分：优13例,良9例,一般1例.结论 股骨近端LCP内固定治疗股骨转子间骨折创伤小、出血少、对骨膜影响小,符合解剖形态,临床疗效满意.     

Advanced Pharmacy Practice Experiences in Pharmacogenomics Offered by US Pharmacy Programs

Objective. To characterize advanced pharmacy practice experiences (APPEs) with a primary focus in pharmacogenomics at schools and colleges of pharmacy in the United States.Methods. This was a cross-sectional, multicenter, observational study of pharmacogenomics APPEs at US pharmacy schools. Directors of experiential education at 146 accredited schools of pharmacy were contacted by phone and asked if their school offered a pharmacogenomics APPE. The preceptors of pharmacogenomics APPEs identified by this phone screen were sent an email with a link to an online survey that asked about their APPE offerings.Results. Of the 142 schools of pharmacy that were successfully reached via phone, 40 (28%) offered an APPE with a primary focus in pharmacogenomics. Thirty unique APPEs with pharmacogenomics as a primary focus were identified. The total number of preceptors involved in the pharmacogenomics APPEs was 33: 19 (58%) faculty preceptors and 14 (42%) non-faculty preceptors. Twenty-three of the 30 pharmacogenomics APPEs completed the survey (77% response rate). The APPE sites were diverse and included academic medical centers, community health systems, pharmacogenomic testing laboratories, and schools of pharmacy. Each pharmacogenomics APPE accommodated an average of six students per year. The APPE activities varied across sites.Conclusion. Only a small number of US pharmacy schools offer an APPE with a primary focus in pharmacogenomics. These rotations are diverse in scope and precepted by faculty or non-faculty pharmacists. The Academy should pursue opportunities to increase experiential education in pharmacogenomics.     

Detection of the hemoglobin E mutation using the color complementation assay: application to complex genotyping

The color complementation assay (CCA) is a method of allele-specific DNA amplification by which competitive priming and extension of fluorescently labeled oligonucleotide primers determine the color of DNA amplification product. This diagnostic method precludes the need for radioisotopes, electrophoresis, and multiple high-stringency reaction conditions. The multiplicity of mutant globin genes present in Southeast Asians complicates clinical diagnosis and underscores the importance of DNA-based diagnostic methods. We have applied CCA to distinguish beta A and beta E alleles. Competing 15mer primers were a fluorescein-labeled complement to beta A and a rhodamine-labeled complement to beta E, identical except for their central nucleotides. A common unlabeled primer was used to amplify DNA product, the color of which was determined by the perfectly complementary primer. Color photography and spectrofluorometry, as well as a method of black-white photography that we developed to distinguish fluorescein- and rhodamine- labeled DNA, were used to record results. We applied CCA to define the complex genotype of a Thai woman with thalassemia intermedia, 96% HbE, and 4% HbF whose possible genotypes included several permutations of alpha-thalassemia, beta-thalassemia, and beta E genes. zeta-Globin gene mapping of DNA doubly digested with Bg/II and Asp 718 showed the -alpha 3.7/--SEA genotype, and CCA confirmed homozygous beta E/beta E. The CCA is useful for diagnosing the compound hemoglobin genotypes of Southeast Asians and could be applied also to prenatal diagnosis in this population.     

Diagnostic role of an immunoassay-detected polymorphism of factor IX for potential carriers of hemophilia B

In hemophilia B, assays based on a monoclonal antifactor IX specific for the Thr-148 variant of an exonic polymorphism have diagnosed carriers in selected families by either establishing linkage or by indicating the presence or absence of a given normal factor IX. The sensitivity of the immunoassays for detecting heterozygous women was explored by comparing results from immunoassays with solid-phase polyclonal v the monoclonal antifactor IXs. Factor IX with the normal Ala-148 variant gave a flat dilution curve, qualitatively distinct from factor IX with the Thr-148 variant in the monoclonal assay. The two were indistinguishable in the polyclonal assay. Mixtures of equal amounts of the two types gave an intermediate result, about half as reactive in the monoclonal as compared with the polyclonal assay system. Whereas mixtures with 10% Ala-148 and 90% Thr-148 factor IXs could not readily be distinguished from Thr-148 factor IX plasma, as little as 1% of the Thr-148 protein was detected in Ala-148 factor IX plasma. The frequency of the Ala-148 variant varied in individuals with different ethnic backgrounds; it was found in 29% of white, 12% of black, and none of Asian blood donors' factor IX genes in Seattle. Only 4% of samples from South African black men were nonreactive (ie, Ala- 148). The Thr/Ala-148 dimorphism is in strong linkage disequilibrium with Taql restriction fragment length polymorphisms (RFLPs). Three recombinations were noted in normal white genes and one in a normal black factor IX gene (less than 2% of those examined). In 34 white families with at least one woman being a possible carrier, genetically, the immunoassay results were informative in 18. RFLP analyses were informative in eight of the 15 families tested. In five families each, assignment of carrier status was made to a woman by only DNA or only immunoassay results, whereas the other approach was noninformative. The immunoassays provide a rapid, inexpensive screening test and complement DNA analysis in white women who are potential carriers of hemophilia B.     

Lessons Learned: Recruiting Research Participants from an Underrepresented Patient Population at a Safety Net Hospital

BackgroundRecruiting participants to clinical research studies is challenging, especially when conducted in safety net settings. We sought to compare the efficacy of different recruitment strategies in an NIH-funded study assessing treatment burden in patients with multiple chronic conditions (MCCs).MethodsTargeted mailing, in-person table-based recruitment (“tabling”) in the waiting room, and telephone calling were used to enroll subjects into one of two studies of treatment burden: a survey study to validate a brief measure of treatment burden for quality assessment (study 1) or a qualitative study to develop a treatment burden clinical communication tool (study 2).ResultsOver 50% of subjects in each study were African American or African immigrants. In study 1, the enrollment goal of 200 was reached within 4 months. Tabling enrolled 78.5% of patients, while the remainder (21.5%) were enrolled from phone calls to eligible patients identified through the electronic medical record (EMR). In study 2, 340 eligible patients were identified through the EMR, and 7 (2.1%) were successfully enrolled via mailed invitations and responses. Retention rates (66% in study 1 and 71% in study 2) were reasonable in all groups.ConclusionsStudy recruiting goals in our safety net population were rapidly reached using the tabling method, which had substantively higher enrollment rates than mailings or telephone calls based on EMR reports. Future trials could compare recruitment strategies across settings and clinical populations.     

胰腺癌组织VEGF和MVD表达与CT灌注成像的关系

血管内皮生长因子作为肿瘤血管生成的一种主要调控因子,与微血管密度密切相关．胰腺癌组织的血管内皮生长因子和微血管密度的测定对于判断患者的预后,指导临床治疗和判断疗效等具有重要作用．CT灌注成像可以通过无创伤检查反映胰腺癌的肿瘤血管构成, 在治疗前后提供有价值依据,对胰腺癌的诊治有重要意义．