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Biomedical subjects

Robert F Vogt

Publications and source records attributed to Robert F Vogt.

10 recordsLinked to original sources
PubMed2006

Comparison of fluorescein and phycoerythrin conjugates for quantifying CD20 expression on normal and leukemic B-cells.

BACKGROUND: Numerous methods for quantitative fluorescence calibration (QFC) have been developed to quantify receptor expression on lymphocytes as potential disease biomarkers. CD20 expression in B-cell chronic lymphocytic leukemia (B-CLL) is one of the best examples of such a biomarker, but results from the use of different QFC methods vary considerably. METHODS: We measured CD20 expression on normal and B-CLL B-cells, using FITC and PE conjugates from the same monoclonal antibody (Mab). As a biological control and calibrator, we also measured CD4 expression on T-cells with FITC and PE Mab. Calibration curves were constructed using the CLSI (formerly NCCLS) consensus guidelines for QFC. Calibration with QuantiBRITE PE-labeled microspheres and the use of unimolar PE conjugates provided direct measurement of antibody bound per cell (ABC) for CD4 and CD20. Calibration for FITC conjugates was based on molecules of equivalent soluble fluorochrome (MESF), as determined by NIST RM 8640 microsphere standards. These MESF values were then converted to ABC, using the CD4 T-cell as a biologic calibrator, to normalize FITC and PE results for CD20 expression. RESULTS: On normal B cells, the mean ABC value for unimolar CD20-PE conjugate was 143,500 (CV +/- 19.1%). The mean ABC value for B-CLL B-cells stained with the same conjugate was 21,700 (CV +/- 42.0%). Using the CD4 T-cell as a biologic calibrator for FITC conjugate, the mean ABC value for CD20-FITC on normal B-cells was 199,300. CD20-FITC staining on B-CLL cells was generally too weak for accurate quantification. On normal T-cells, the mean ABC value for CD4 unimolar PE conjugate was (36,800 +/- 10.4)%, and it did not differ significantly in CLL samples. CONCLUSION: The expression of CD20 on normal and B-CLL lymphocytes can be quantified in ABC units using unimolar CD20-PE conjugates. In addition, CD4 expression on T-cells can be used as a biological calibrator to quantify CD20-FITC ABC, with reasonable agreement between the two conjugates with different fluorochromes. Issues regarding the accuracy of MESF microsphere calibrators and effective F/P ratios for FITC conjugates will require additional laboratory studies.

Antigens, CD20↗
PubMed2006

A screening system for detecting genetic risk markers of type 1 diabetes in dried blood spots.

BACKGROUND: Certain alleles among the genes that code for the human leukocyte antigens (HLA) confer susceptibility or resistance to the development of autoimmunity that causes type 1 diabetes (T1D). A number of ongoing diabetes research studies analyze dried blood spots (DBS) from newborn infants for HLA-D alleles to identify higher-risk children as early as possible. A commercially available assay to detect such alleles has recently become available using a dissociation- enhanced lanthanide fluorescence system found in many newborn screening laboratories. METHODS: We adapted the system for use with DBS and improved the sample set-up for greater efficiency. We also developed an independent system for data analysis based on a spreadsheet program. These modifications were applied to HLA-DQB1 gene locus (DQB) analysis of 117 newborn DBS, and the results we obtained were compared with independent reference values. RESULTS: Our assay modifications and independent data analysis improved sample throughput and result tabulation. DQB results from the modified assay were consistent with the reference values in all but one sample, which showed a partial match. CONCLUSIONS: The modifications described here make this commercially available assay more suitable for high-throughput applications such as newborn screening. Our results show that this system allows highly accurate detection of DQB alleles that influence T1D risk.

Blood Chemical Analysis↗
PubMed2004

Applying public health strategies to primary immunodeficiency diseases: a potential approach to genetic disorders.

Primary immunodeficiency (PI) diseases are a group of primarily single-gene disorders of the immune system. Approximately 100 separate PI diseases have been described, but <20 probably account for >90% of cases. Although diverse, PI diseases share the common feature of susceptibility to infection and result in substantial morbidity and shortened life spans. Most important, prompt diagnosis and treatment can now lead to life-saving treatment and result in marked improvements in the quality and length of life for persons with PI diseases. In November 2001, a workshop was convened by CDC in Atlanta, Georgia, to discuss ways to improve health outcomes among persons with PI disease. A multidisciplinary panel of persons knowledgeable in PI diseases and public health met to identify and discuss public health strategies that can be applied to PI diseases and possibly for other genetic disorders. A systematic assessment based on the established public health framework was applied to the growing group of PI diseases, whose diverse genetic mutations span multiple components of the immune system but all lead to increased incidence and severity of infections. During the meeting, specialists in clinical immunology, public health, genetics, pediatrics, health communication, and ethics from state and federal agencies, academic centers, professional organizations, and advocacy foundations discussed the four components of the public health framework as they relate to PI diseases. These four components include 1) public health assessment (application of traditional public health methods to assess the occurrence and impact of PI diseases on communities); 2) population-based interventions (development, implementation, and evaluation of screening tests administered to newborns and clinical algorithms for early recognition of symptomatic persons to facilitate the earliest possible diagnosis and treatment for PI diseases); 3) evaluation of screening and diagnostic tools (to ensure their quality and appropriateness for identification of patients with PI diseases); and 4) communication (communication with and information dissemination to health-care providers and the public to facilitate prompt and appropriate diagnosis and intervention). The working group's deliberations focused on challenges and opportunities, priority research questions, and recommendations for future action for these four components. These recommendations, developed by workshop participants, will be useful to medical and public health professionals who are evaluating methods to increase recognition of PI diseases and other genetic disorders.

Adolescent↗
PubMed2004

Formalization of the MESF unit of fluorescence intensity.

This report summarizes the work performed during the past two years at the National Institute of Standards and Technology (NIST) in the refinement and formal definition of the MESF unit of fluorescence intensity. In addition to the theory underlying the MESF unit, considerations of error analysis are also presented. The details of this work may be found in the three publications of the NIST Journal of Research (www.nist.gov) listed as the references 2-4. The use of the fluorescence intensity unit provides a tool to compare quantitative fluorescence intensity measurements over time and across platforms.

Antibodies, Monoclonal↗
PubMed2003

B-cell monoclonal lymphocytosis and B-cell abnormalities in the setting of familial B-cell chronic lymphocytic leukemia.

BACKGROUND: Among all hematologic malignancies, B-cell chronic lymphocytic leukemia (BCLL) has the highest familial clustering (three- to sevenfold increase), strongly suggesting a genetic component to its etiology. Familial BCLL can be used as a model to study the early pathogenesis of this disease. METHODS: We examined nine kindreds from the National Cancer Institute's Familial BCLL Registry, consisting of 19 affected members with BCLL and 33 clinically unaffected first-degree relatives. Flow cytometric immunophenotyping to detect a B-cell monoclonal lymphocytosis (BCML) was performed. Monoclonality was confirmed by polymerase chain reaction analysis of whole blood DNA. Cell cycle analysis for aneuploidy was conducted. RESULTS: In all affected individuals, we observed the classic BCLL CD5/CD19/CD20/CD23 immunophenotypic patterns. Six of the 33 unaffected individuals (18%) had evidence of BCML. Additional individuals (13/33, 39%) showed some other abnormality, whereas 14 individuals (42%) were normal. Based on an estimated prevalence of 0.7% for BCML in the general population, the finding of six subjects (18%) with clonal abnormalities in this relatively modest sample was significantly greater than expected (i.e., 18% vs. 0.7%, P < 5.7 x 10(-9)). CONCLUSIONS: Individual components of BCML and other B-cell abnormalities were observed in almost half of the apparently unaffected individuals. Our findings suggested that BCML may be an early detectable abnormality in BCLL. The spectrum of some of these observed abnormalities suggested the involvement of different B-cell subpopulations or different pathways in clonal evolution. Population-based, longitudinal studies will be required to determine the incidence of BCML and other B-cell abnormalities and their relation to disease progression in BCLL and other closely related B-cell lymphoproliferative disorders.

B-Lymphocytes↗
PubMed2003

Reproducibility of immunological tests used to assess multiple chemical sensitivity syndrome.

Whether persons with multiple chemical sensitivity syndrome (MCS) have immunological abnormalities is unknown. To assess the reliability of selected immunological tests that have been hypothesized to be associated with MCS, replicate blood samples from 19 healthy volunteers, 15 persons diagnosed with MCS, and 11 persons diagnosed with autoimmune disease were analyzed in five laboratories for expression of four T-cell surface activation markers (CD25, CD26, CD38, and HLA-DR) and in four laboratories for autoantibodies (to smooth muscle, thyroid antigens, and myelin). For T-cell activation markers, the intralaboratory reproducibility was very good, with 90% of the replicates analyzed in the same laboratory differing by < or = 3%. Interlaboratory differences were statistically significant for all T-cell subsets except CD4+ cells, ranging from minor to eightfold for CD25+ subsets. Within laboratories, the date of analysis was significantly associated with the values for all cellular activation markers. Although reproducibility of autoantibodies could not be precisely assessed due to the rarity of abnormal results, there were inconsistencies across laboratories. The effect of shipping on all measurements, while sometimes statistically significant, was very small. These results support the reliability of fresh and shipped samples for detecting large (but perhaps not small) differences between groups of donors in the T-cell subsets tested. When comparing markers that are not well standardized, it may be important to distribute samples from different study groups evenly over time.

Antigens, Differentiation, T-Lymphocyte↗
PubMed2002

Report from a workshop on multianalyte microsphere assays.

Multiplexed assays using fluorescent microspheres is an exciting technique that has been gaining popularity among researchers, particularly those in the public health field. Part of its popularity is due to its flexibility, as both immunoassays and oligonucleotide hybridization assays can be developed on this platform. This report summarizes a workshop held by the Centers for Disease Control and Prevention that discussed issues surrounding these assays and the Luminex 100 xMAP instrument. Topics included instrumentation, assay design, sample matrix and volume, quality control, and development of commercial applications.

Animals↗
PubMed2002

Frequency-domain measurement of the photodegradation process of fluorescein.

The frequency-domain technique is applied to measure the photodegradation rate of fluorescein in aqueous solutions. The illuminating light is modulated, and the changes in fluorescence from the illuminated region are detected synchronously. A constant flow rate is imposed on the fluorescein solution to control the mass transport of fluorescein into the illuminated region. The fluorescence response is described by a model that assumes that photodegradation occurs from the triplet excited state. The predictions of the model are consistent with the observed variations in the fluorescence response with flow rate, modulation frequency and incident power. We discuss in this article how the dependence of the model parameters on experimental conditions can be used to infer the photodegradation rate as well as some of the details of the photodegradation mechanism. The results are consistent with the known mechanism of photodegradation of fluorescein. The frequency-domain technique gives a photodegradation rate of 53 s(-1) in an air-saturated solution and 37 s(-1) in solutions purged with argon gas.

Journal Article↗