Biomedical subjects

Jose L Matud

Publications and source records attributed to Jose L Matud.

4 recordsLinked to original sources

PubMed2007

Assessing immunophenotyping performance: proficiency-validation for adopting improved flow cytometry methods.

BACKGROUND: The continuous improvement and evolution of immune cell phenotyping requires periodic upgrading of laboratory methods and technology. Flow cytometry laboratories that are participating in research protocols sponsored by the NIAID are required to perform "switch" studies to validate performance before methods for T-cell subset analysis can be changed. METHODS: Switch studies were conducted among the four flow cytometry laboratories of the Multicenter AIDS Cohort Study (MACS), comparing a 2-color, lyse-wash method and a newer, 3-color, lyse no-wash method. Two of the laboratories twice failed to satisfy the criteria for acceptable differences from the previous method. Rather than repeating more switch studies, these laboratories were allowed to adopt the 3-color, lyse no-wash method. To evaluate the impact of the switch to the new method at these two sites, their results with the new method were evaluated within the context of all laboratories participating in the NIH-NIAID-Division of AIDS Immunology Quality Assurance (IQA) proficiency-testing program. RESULTS: Laboratory performance at these two sites substantially improved relative to the IQA standard test results. Variation across the four MACS sites and across replicate samples was also reduced. CONCLUSIONS: Although switch studies are the conventional method for assessing comparability of laboratory methods, two alternatives to the requirement of repeating failed switch studies should be considered: (1) test the new method and assess performance on the proficiency testing reference panel, and (2) prior to adoption of the new methods, use both the old and the new method on the reference panel samples and demonstrate that performance with the new method is better according to standard statistical procedures. These alternatives may help some laboratories' transition to a new and superior methodology more quickly than if they are required to attempt multiple, serial switch studies.

Acquired Immunodeficiency Syndrome↗

PubMed2005

Effects of HIV-1 infection on lymphocyte phenotypes in blood versus lymph nodes.

Most immunopathogenesis studies of HIV-1 use peripheral blood. Most lymphocytes reside in lymphoid tissues, however, and the extent to which blood mirrors tissues is unclear. Here, we analyze lymphocytes in blood and lymph nodes of HIV-1-uninfected and -infected persons. Baseline comparison of node and blood lymphocytes in seronegative persons demonstrates a lower ratio of CD8+ versus CD4+ T lymphocytes, a lower number of effector cells (CD28-) within the CD8+ compartment, and greater activation (D-receptor [DR+]) within the CD4+ compartment. In infected versus uninfected persons, nodes exhibit elevated CD8+ T lymphocytes with an increased memory-effector phenotype (CD62L-/CD45RA-) and activation (CD38+ and DR+) but minimal differences in the CD4+ compartment. Changes attributable to HIV-1 infection are markedly greater in node lymphocytes than in blood. Comparisons of CD8+ T-lymphocyte parameters and viremia in infected persons reveal positive correlations of CD38+ expression on cells in blood and nodes and a negative correlation of terminal effector cells (CD62L-/CD45RA+) in the nodes to viremia. Multiple linear regression analysis indicates that CD38 expression on node (not blood) CD8+ T lymphocytes is the sole independent predictor for viremia. Thus, blood indirectly reflects processes in lymphoid tissues, and caution should be applied when interpreting immunopathogenesis studies of blood.

Adult↗

PubMed2002

Simultaneous flow cytometric analysis of two cell surface markers, telomere length, and DNA content.

BACKGROUND: Various protocols for estimation of telomere length in individual cells by flow cytometry using fluorescence in situ hybridization of fluorescently labeled peptide nucleic acid (PNA) probes (Flow-FISH) have been described. Combined analysis of telomere length and cell phenotype, however, remains difficult because few fluorochromes with suitable emission spectra tolerate the harsh conditions needed for DNA denaturation during hybridization of the telomere-specific PNA probe. We overcame these problems and developed a method for measuring telomere length in cell subsets characterized by the expression of two surface antigens. METHODS: Alexa Fluor 488 and Alexa Fluor 546 were used for cell surface staining. Antigen-antibody complexes were covalently cross-linked onto the cell membrane before Flow-FISH. Cells were hybridized with a PNA probe conjugated to cyanine 5 (Cy5). Hoechst 33342 (HO342) was added for determination of cellular DNA content. For assay standardization, we added an aliquot of a single batch of 1,301 cells to each sample as an internal control before hybridization with the PNA probe. Samples were prepared in duplicate and analyzed on a standard three-laser BD LSR flow cytometer. For assay validation, the same samples were analyzed in parallel to correlate the percentage of telomere length of the sample versus 1,301 control cells to the mean size of terminal restriction fragments (TRFs) of DNA as determined by Southern gel analysis. RESULTS: The method permitted clear identification of lymphocyte subsets in samples hybridized for Flow-FISH, with subset frequencies comparable to those of untreated samples. At a concentration of 10 nM, the Cy5-labeled telomere-specific PNA probe produced a bright fluorescence signal well separated from background. Addition of HO342 in low concentration did not interfere with Cy5 telomere fluorescence, produced adequate DNA histograms, and permitted clear identification of cell phenotype. The probe concentration of 10 nM also proved optimal for inclusion of 1,301 control cells for assay standardization. Telomere length estimations by the current method correlated highly with TRF calculations by Southern gel hybridization (r(2)= 0.9, P = 0.0003). Application of our protocol to the analysis of human CD8CD28 lymphocyte subsets showed that CD8(+bright)CD28(-) lymphocytes generally exhibit shorter telomeres than CD8(+bright)CD28(+) cells. These data concurred with previous results of telomere shortening in CD8(+)CD28(-) T cells that were obtained by using different techniques. CONCLUSIONS: The multiparameter Flow-FISH protocol permitted rapid determination of differences in telomere length in subpopulations characterized by two surface markers without prior cell separation.

Antigens, CD↗

PubMed2002

Predictive value of immunologic and virologic markers after long or short duration of HIV-1 infection.

Laboratory markers that predict HIV-1 disease progression include plasma viral burden, CD4+ T-cell count, and CD38 expression on CD8 T cells. To better understand whether the predictive value of these markers is dependent on how long an individual has been infected, we analyzed data from the Multicenter AIDS Cohort Study early (median = 2.8 years) and late (median = 8.7 years) in the course of infection. Overall, we found that HIV RNA and CD38 levels were similarly predictive of AIDS early on compared with a relatively weaker CD4 cell count signal. Later in the course of infection, CD38 level remained the strongest predictive marker and CD4 cell count registered a marked increase in prognostic power. Among untreated individuals, there was little difference in prognosis (median time to AIDS) associated with given marker values regardless of infection duration. The prognosis given a specific viral load level tended to deteriorate late in the course of infection among those undergoing treatment with monotherapy or combination therapy, however. These data provide a unique historical look at the predictive value and prognostic significance of HIV-1 disease markers at different stages of infection in a large cohort, with direct relevance to current patients who are untreated or for whom treatment is ineffective.

ADP-ribosyl Cyclase↗