Laboratory of Stem Cell Biology

Bethesda, MD, United States

Laboratory of Stem Cell Biology

Bethesda, MD, United States
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Li H.-F.,Laboratory of Stem Cell Biology | Li H.-F.,Sichuan University | Meng W.-T.,Laboratory of Stem Cell Biology | Meng W.-T.,Sichuan University | And 12 more authors.
Medicine (United States) | Year: 2016

B cell acute lymphoblastic leukemia (B-ALL) exhibits phenotypes reminiscent of normal stages of B-cell development. As demonstrated by flow cytometry, the immunophenotypes are able to determine the stages of B cell development. Multicolor flow cytometry (MFC) is more accurate at identifying cell populations. In this study, 9-color panels, including CD10, CD19, CD20, CD22, CD34, CD79a, CD179a, and IgM, which are sequentially expressed during B cell development, were designed to detect the leukemia cell subpopulations in adult B-ALL patients. In 23 patients at diagnosis, 192 heterogeneous subpopulations of leukemia cells were detected. Compared with their counterparts at diagnosis and after the 1st course of induction therapy, the responses of the subpopulations were also heterogeneous. In the CD10+ population, the residual B cell subpopulations in the BCR/ABL+ patients were obviously reduced compared to those in the BCR/ABL- patients. New subpopulations were detected in 22 of 23 patients and were primarily located in the CD34+CD10- populations. Subpopulations of clonal evolution were heterogeneous after induction therapy. Our results suggest that the subpopulations in B-ALL patients should be dynamically monitored by development-Associated immunophenotyping before, during, and after induction therapy and to predict the prognosis of the disease. © Copyright 2016 the Author(s). Published by Wolters Kluwer Health, Inc. All rights reserved.

Wang L.,U.S. National Institute of Standards and Technology | Abbasi F.,Laboratory of Stem Cell Biology | Jasper G.A.,U.S. National Institutes of Health | Kreitman R.J.,U.S. National Institutes of Health | And 3 more authors.
Cytometry Part B - Clinical Cytometry | Year: 2011

Background: Quantitative flow cytometry (QFCM) is being applied in the clinical flow cytometry laboratory. Quantitative normal T cell CD4 expression represents a biologic standard and quality control agent. However, low levels of CD4 expression were detected in normal T cells in Hairy Cell Leukemia (HCL) samples. Methods: The QuantiBrite System® was used to determine the level of CD4 expression (mean antibody bound per cell, ABC) in fresh and shipped HCL blood and fresh normal donor blood (NDB). The effects of shipping, lysing reagent, cell preparation method, and antibody lot were evaluated. Results: Shipped HCL specimens (n = 69) had a significantly lower mean CD4 ABC of 38,788 (CV = 9.1%) compared to fresh specimens (n = 105) CD4 value of 40,330 (CV = 8.4%) (P < 0.05). In NDB, significant differences were seen for fresh versus shipped specimens using the stain/lyse method but not for lyse/stain method. Consistent differences in CD4 ABC based upon antibody lot were observed in fresh HCL and NDB samples. Stain/lyse and lyse/stain methods using NH 4Cl lyse were compared in NDB using identical samples and antibodies. The NDB CD4 ABC values obtained with the lyse (NH 4Cl)/stain method (45,562, 3.7% CV) were lower than those obtained with the stain/lyse (NH 4Cl) method (49,955, 3.3% CV) with P < 0.001. Conclusions: CD4 expression in HCL patient samples is not inherently different from that observed in NDB and therefore may serve as a biological control in clinical QFCM. Technical variables impact significantly on QFCM of CD4. Published 2010 Wiley-Liss, Inc. © Published 2010 Wiley.

Wang M.,University of Maryland College Park | Wang M.,U.S. National Institute of Standards and Technology | Misakian M.,U.S. National Institute of Standards and Technology | He H.-J.,U.S. National Institute of Standards and Technology | And 7 more authors.
Clinical Proteomics | Year: 2014

Background: In our previous study that characterized different human CD4+ lymphocyte preparations, it was found that both commercially available cryopreserved peripheral blood mononuclear cells (PBMC) and a commercially available lyophilized PBMC (Cyto-Trol™) preparation fulfilled a set of criteria for serving as biological calibrators for quantitative flow cytometry. However, the biomarker CD4 protein expression level measured for T helper cells from Cyto-Trol was about 16% lower than those for cryopreserved PBMC and fresh whole blood using flow cytometry and mass cytometry. A primary reason was hypothesized to be due to steric interference in anti- CD4 antibody binding to the smaller sized lyophilized control cells.Method: Targeted multiple reaction monitoring (MRM) mass spectrometry (MS) is used to quantify the copy number of CD4 receptor protein per CD4+ lymphocyte. Scanning electron microscopy (SEM) is utilized to assist searching the underlying reasons for the observed difference in CD4 receptor copy number per cell determined by MRM MS and CD4 expression measured previously by flow cytometry.Results: The copy number of CD4 receptor proteins on the surface of the CD4+ lymphocyte in cryopreserved PBMCs and in lyophilized control cells is determined to be (1.45∈±∈0.09)∈×∈105 and (0.85∈±∈0.11)∈×∈105, respectively, averaged over four signature peptides using MRM MS. In comparison with cryopreserved PBMCs, there are more variations in the CD4 copy number in lyophilized control cells determined based on each signature peptide. SEM images of CD4+ lymphocytes from lyophilized control cells are very different when compared to the CD4+ T cells from whole blood and cryopreserved PBMC.Conclusion: Because of the lyophilization process applied to Cyto-Trol control cells, a lower CD4 density value, defined as the copy number of CD4 receptors per CD4+ lymphocyte, averaged over three different production lots is most likely explained by the loss of the CD4 receptors on damaged and/or broken microvilli where CD4 receptors reside. Steric hindrance of antibody binding and the association of CD4 receptors with other biomolecules likely contribute significantly to the nearly 50% lower CD4 receptor density value for cryopreserved PBMC determined from flow cytometry compared to the value obtained from MRM MS. © 2014 Wang et al.; licensee BioMed Central.

Marti G.E.,Laboratory of Stem Cell Biology | Stetler-Stevenson M.,Laboratory of Stem Cell Biology | Grant N.D.,Laboratory of Stem Cell Biology | White T.,Laboratory of Stem Cell Biology | And 7 more authors.
Leukemia and Lymphoma | Year: 2011

This is a phase I study of 7-hydroxystaurosporine (UCN-01) and fludararbine monophosphate (FAMP) in relapsed lymphoma. UCN-01 alone was administered in cycle 1 and with FAMP in cycles 26. FAMP was escalated in cohorts from 1 to 5 days. UCN-01 and FAMP pharmacokinetics and apoptosis of malignant lymphocytes was evaluated. Eighteen patients were enrolled. Standard FAMP with UCN-01 was tolerated without dose-limiting toxicity (DLT) and those seen were common to either agent alone. One patient died due to Stevens-Johnson syndrome. Seven of 18 patients responded. No pharmacological effect of UCN-01 by FAMP was noted. Lymphocytosis occurred in 15 of 18 patients following UCN-01 to paradoxically increase circulating tumor cells. UCN-01 induced apoptosis in six of eight patients with chronic lymphocytic leukemia (CLL). UCN-01 does not increase FAMP toxicity. Transient lymphocytosis followed by apoptosis occurs with UCN-01. Mobilization from tissue reservoirs may play a role in the induction of cell death in malignant lymphocytes. © 2011 Informa UK, Ltd.

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