PubMed Health⌕ Search

Biomedical subjects

D Printz

Publications and source records attributed to D Printz.

At least 37 records · Page 2Linked to original sources
PubMed1999

Kinetics of chimerism during the early post-transplant period in pediatric patients with malignant and non-malignant hematologic disorders: implications for timely detection of engraftment, graft failure and rejection.

The monitoring of chimerism by PCR has become a routine diagnostic approach in patients after allogeneic bone marrow or peripheral blood stem cell transplantation. Nevertheless, a temporal correlation between molecular and hematologic assessment of engraftment has not been clearly established. To address this issue, and to determine the potential clinical implications of early kinetics of mixed chimerism, we have investigated 66 allogeneic stem cell transplantations (SCTs) in 58 pediatric patients suffering from different types of leukemia (n = 44) or non-malignant hematologic disorders (n = 14) by close molecular monitoring during the first days and weeks after transplantation. Patient- and donor-derived hematopoiesis were assessed at 1- to 3-day intervals in peripheral blood samples by PCR analysis of highly polymorphic microsatellite loci (STR-PCR). Detection of an increasing, and ultimately dominant donor-specific allelic pattern, which we defined as molecular engraftment, preceded hematologic engraftment by a median of 7 days (range 1-17 days) in all patients investigated. PCR analyses during the first days after transplantation facilitated detection of molecular engraftment according to the above definition by day +14 (range day +2 to day +14), thus permitting prediction of successful engraftment (upper limit of the two-sided confidence interval po = 6%) while the peripheral leukocyte counts were mostly below 200/microl. In three cases, however, the criteria for molecular engraftment were not fulfilled by day +14. These patients also failed to show hematologic engraftment, and required a second transplantation. Close monitoring by STR-PCR showed that graft rejection and autologous recovery can occur early and with very rapid dynamics. Molecular analysis of specific leukocyte subsets isolated by flow-sorting enabled sensitive assessment of changes in the pattern of chimerism which had escaped detection in assays using whole white blood cell (WBC) samples. This approach facilitated the identification of expanding or decreasing recipient cells, and permitted early detection of impending rejection or relapse. Moreover, monitoring of the dynamics of chimerism allowed rapid assessment of the response to therapy. Our observations provide support for the concept of initiating genotype analyses early after SCT and monitoring at rather short intervals to permit timely evaluation of clinically relevant processes, and to provide a basis for early implementation of treatment.

Adolescent↗
PubMed1998

Four-color flow cytometric investigation of terminal deoxynucleotidyl transferase-positive lymphoid precursors in pediatric bone marrow: CD79a expression precedes CD19 in early B-cell ontogeny.

Terminal deoxynucleotidyl transferase (TdT)-positive cells in human bone marrow (BM) are a phenotypically inhomogeneous population of precursor cells. In their majority, these TdT+ cells are unambiguously committed to the B lineage, as evidenced by CD19 expression. However, TdT+ precursors that lack CD19 also exist and these may encompass a differentiation potential for the B as well as for other lineages. Because recent data suggested that CD19 expression is not the earliest differentiation event in B-cell ontogeny, we sought to reevaluate TdT+ lymphoid precursors in pediatric BM to define the phenotypic denominator of B-lineage affiliation upstream of CD19. Using four-color flow cytometry, we focused on the assessment of the CD79a antigen, which is highly B-cell specific and which may also be expressed very early in B-cell ontogeny. We found that a majority of TdT+ cells coexpressed CD19 and CD79a in addition to CD10 and CD34, whereas, in all investigated samples, some TdT+ precursors lacked CD19 but expressed CD79a, which suggestively indicates also their B-lineage affiliation. In contrast to the CD19(+) precursors, which were usually CD10(hi) and CD79b+, these CD19(-)CD79a+ putative B-cell precursors preferentially expressed CD10 at low levels and were CD79b+ in only 41%. About 17% of these TdT+CD19(-)CD79a+ precursors also coexpressed CD33 and CD7, but not myeloperoxidase, CD14, or cytoplasmic CD3, which is discussed in the light of cellular activation rather than lineage promiscuity. Our data confirm that the earliest differentiation stages of B cells can be dissected upon expression of the lineage antigens CD79a and CD19 and imply that CD79a is earlier expressed than CD19. This raises the chance to follow the sequential events heralding B-cell commitment in the most immature precursors by correlating phenotypic and genetic differentiation markers.

Antigens, CD↗
PubMed1998

A novel bioassay for P-glycoprotein functionality using cytochalasin D.

The functional contribution of both P-glycoprotein (P-gp) and the multidrug resistance-associated protein (MRP) to multidrug resistance (MDR) in tumor cells is commonly determined by drug cytotoxicity and/or accumulation/efflux tests. We report on a bioassay developed for the specific detection of functional P-gp levels and the efficacy of related chemosensitizers (CD-P-gp-assay). The assay is based on the flow cytometric measurement of changes in the > or = G2M cell cycle compartment which are due to the induction of polykaryons after exposure of proliferating cells to three defined cytochalasin D (CD) concentrations with and without verapamil. As demonstrated in 13 well-characterized MDR cell models (20 resistant sublines), there is a significant correlation between cytokinesis-blocking CD doses, as well as responsiveness to chemosensitizers and MDR1 gene expression (mRNA and P-gp) allowing discrimination between different levels of P-gp-MDR. CD-P-gp-assay specificity was assessed by testing 23 compounds: 19 known as potent inhibitors of P-gp-MDR, some of them, though to a lesser extent, also of MRP-MDR; 1 inhibiting MRP-but not P-gp-MDR; 3 inactive in both types of MDR. A modulation of CD activity was confined exclusively to both P-gp-expressing cell lines and P-gp chemosensitizers. CD cytoskeletal activity measured by FACS is a specific and sensitive tool with which to detect functional P-gp and related chemosensitizers.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Comparative phenotype mapping of normal vs. malignant pediatric B-lymphopoiesis unveils leukemia-associated aberrations.

Leukemic cells of B-lineage acute lymphoblastic leukemia (ALL) are regarded as the malignant counterparts of immature, physiologic B cell precursors (BCPs). To determine whether phenotypic differences exist between these corresponding cell types, we investigated samples of normal pediatric bone marrow (n=30) as well as of B-precursor ALL at diagnosis (n=53; common and pre-B subtype). Using three-color multiparameter flow cytometric analysis, we compared the leukemic populations with the physiologic BCPs of corresponding maturity with respect to the intensity with which they expressed a series of antigens. In some of these antigens, leukemia-associated aberrations were frequently observed. In particular, overexpression of CD10 was displayed by 65% of ALL samples, whereas 58% of leukemic cases aberrantly exhibited very low or no CD45RA expression. Regarding CD11a and CD44, 47% and 35% of ALL populations were aberrant as defined by either the absence or significant overexpression of the antigen. In contrast, antigen densities of CD49d, CD49e, and CD99 on leukemic cells were in the normal range of values for BCPs. Combining the patterns of frequently aberrant markers in a comprehensive analysis, we were able to identify individual phenotypic leukemic cell aberrations in up to 98% of investigated cases. CD10 and/or CD45RA were aberrant in 86% of cases overall, emphasizing the high discriminative potential of these two markers. Using comparative phenotype mapping based on quantitatively aberrant, leukemia-associated antigenic patterns, we were able to detect leukemic blasts among normal bone marrow cells at frequencies as low as 10(-5). We speculate that our approach may have a profound impact on the development of new strategies for minimal residual disease investigations in patients with BCP-ALL.

Adolescent↗
PubMed1997

Acute leukemias express a functional receptor for the human growth hormone.

The potential influence of the human growth hormone (hGH) on the behavior of acute leukemias is a matter of controversy. We investigated primary childhood and adult leukemias (n = 44) and leukemic cell lines (n = 13) for the expression of the hGH receptor (hGHR) by immunohistochemistry and flow cytometry. All leukemias expressed the hGHR in the cytoplasm; expression on the surface was undetectable in some of the leukemias. In leukemic cell lines, hGHR expression on the surface was demonstrated in a dose-dependent manner after incubation with rhGH. Physiologic concentrations of hGH were more efficient than higher doses in increasing hGHR surface expression. A proliferative response to hGH was accomplished in cell lines REH, Molt4, and K562. However, only one of 19 primary leukemias (ALL, n = 12; AML, n = 7) showed increased cell counts after the addition of 50-800 ng/ml recombinant hGH (rhGH). These cells were of an immature T-cell phenotype. We thus conclude that acute leukemias can be stimulated by hGH to up-regulate its receptor, but that most primary leukemias may require additional signals for the induction of proliferation.

Adult↗
PubMed1997

Multiparameter phenotype mapping of normal and post-chemotherapy B lymphopoiesis in pediatric bone marrow.

We studied the differentiation profiles of B cell precursors (BCP) in normal and post-chemotherapy pediatric bone marrow (BM) using multiparameter flow cytometry. The goal of our study was to draw a comprehensive phenotypic map of the three major maturational BCP stages in BM. By correlating lineage-associated markers, CD45RA, and several adhesion molecules, the stage-specific patterns were found to differ in certain details from previously published concepts. Among the earliest BCP, a subset of CD34+ CD10(lo) precursors was repeatedly observed in addition to the well characterized CD34+ CD10(hi) CD19+ majority of cells. Only two-thirds of these CD34+ CD10(lo) cells expressed CD19. However, uniformity of phenotypic features, absence of T lineage markers, and the regeneration kinetics after chemotherapy suggest the B lineage affiliation of the CD34+ CD10(lo) precursors in general. In the more mature BCP, expression of CD10, CD20, cytoplasmic and surface mu chains (c mu and s mu) was observed to overlap more than previously recognized. We found that CD20 and c mu appear early during B cell ontogeny (already on CD34+ BCP), and that CD10 is lost late, following the onset of s mu expression. Differences between normal and post-chemotherapy BM specimens regarding the phenotypic appearance of BCP were exclusively due to differences in the subset composition, as post-chemotherapy samples showed a preponderance of immature stages. Our observations may build a framework for comparing leukemic cells with their normal counterparts to define possible leukemia-associated aberrations useful for residual disease studies.

Adolescent↗
PubMed1997

Quantification of CD34+ cells: comparison of methods.

BACKGROUND: Quantification of CD34+ stem and progenitor cells is predominantly performed by flow cytometric analysis of cells prepared by whole blood staining and red cell lysis. This method also includes cell washing, which is thought to cause the destruction and loss of some of the nucleated cells (NCs). To address this cell loss and its influence on the outcome of enumeration, three techniques for preparing cells for quantification of CD34+ cells were compared. STUDY DESIGN AND METHODS: Blood (n = 179), bone marrow (n = 60), and leukapheresis components (n = 64) were examined by the use of density separation of mononuclear cells (MNCs) and two red cell-lysis procedures (wash and no-wash). Cell counts were determined in the original materials and after cell preparation. Absolute CD34+ cell counts were calculated using the flow cytometry-analyzed proportions of CD34+ cells and the various white cell counts. RESULTS: Depending on the cell source and the cell preparation chosen, the loss of NCs ranged between 12 percent and 89 percent of the original white cell number. This loss of NCs was exclusively due to cell washing and predominantly affected granulocytic cells. Analysis of the flow cytometry data revealed that the relative CD34+ values in blood and bone marrow were roughly threefold higher in density separated MNCs than in those that underwent the lyse-and-wash procedure. Calculation of absolute CD34+ cell counts confirmed that the MNC procedure underestimated the CD34+ cell content by a median of 26 percent (blood), 21 percent (bone marrow), and 5 percent (leukapheresis component) when compared with the median yield from analysis and cell counting performed after the lyse-and-wash procedure. On the other hand, the conventional lysis procedure, which applies the original white cell counts for CD34+ quantification, was shown to overestimate the CD34+ cell content by a median of 1.2-fold, 1.33-fold, and 1.13-fold, respectively. CONCLUSION: Neither density separation nor the whole-blood lysis procedure seems appropriate for optimal CD34+ quantification.

Ammonium Chloride↗
PubMed1997

Spiking fevers with clozapine treatment.

Clozapine often causes low-grade fever and less frequently spiking fever. We describe three cases of spiking fever that occurred in the first 3 weeks of clozapine therapy. A new set of side effects of clozapine is identified, which includes spiking fever, respiratory and gastrointestinal symptoms, and neutrophilia. Possible mechanisms are discussed.

Adult↗
PubMed1996

Role of ploidy, chromosome 1p, and Schwann cells in the maturation of neuroblastoma.

BACKGROUND: Neuroblastoma is a heterogeneous disease, with manifestations ranging from spontaneous regression to lethal spread. Sometimes the tumor spontaneously differentiates toward a benign ganglioneuroma (maturing neuroblastoma). The prognosis is frequently related to ploidy, deletions in the short arm of chromosome 1, and amplifications of the N-myc oncogene. Maturing neuroblastomas consist of both neuronal cells and Schwann cells. We investigated the genetic composition of both cell types in maturing neuroblastomas, to determine the relation between genetic abnormalities and maturation. METHODS: We studied 20 maturing and mature neuroblastomas by in situ hybridization to count the chromosomes and evaluate possible deletions in the short arm of chromosome 1 in neuronal and Schwann cells. The DNA content of the cells was measured by flow cytometry. RESULTS: Neuroblastic and ganglionic cells showed aberrations in the number of chromosomes. In situ hybridization and flow cytometry demonstrated near-trip-loidy in 18 of 19 tumors and pentaploidy in the remaining tumor. The Schwann cells in all 20 neuroblastomas contained normal numbers of chromosomes. In 18 tumors studied, there were no chromosome 1 deletions in either type of cell. CONCLUSIONS: The Schwann cells in maturing neuroblastomas differ genetically from the neuronal cells. The normal number of chromosomes in Schwann cells and the abnormal number in neuroblastic ganglionic cells suggests that Schwann cells are a reactive population of normal cells that invade the neuroblastoma. Near-trip-loidy of neuroblastoma cells and intact chromosome 1 are presumably genetic prerequisites for spontaneous organoid maturation, because we found no diploidy or chromosome 1 depletions in the neuronal cells of spontaneously maturing neuroblastomas.

Child↗
PubMed1996

Lack of DNA synthesis among CD34+ cells in cord blood and in cytokine-mobilized blood.

Flow cytometric DNA analysis was performed in combination with three-colour immunological staining of cell surface antigens on density-separated mononuclear cells (MNC) obtained from peripheral blood (PB) before, during and after cytokine stimulation of healthy adults. The aim of the study was to determine the cell-cycling status of haemopoietic progenitor cells mobilized into the blood of healthy volunteers during a 5 d treatment period with 5/micrograms per kg body weight of either granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony-simulating factor (GM-CSF). Despite considerably increasing numbers of CD34+ PB MNC, the latter were not found to be in S/G2M phase, whereas, among the CD34- MNC, the proportion of cells in S/G2M phase increased from < 0.1% to 0.75 +/- 0.4% (GM-CSF) and to 1.34 +/- 0.75% (G-CSF) and dropped again after discontinuation of the cytokine stimulation. These cells expressed CD33 but were negative for CD45RA, CD3, CD19 and CD14 and were thus considered granulopoietic cells. Analogous results were obtained from analyses of cord blood (CB). In contrast, CD34+ cells from bone marrow (BM) were partially (between 9% and 15%) found to be in S/G2M phase. The non-cycling status of PB and progenitor cells was confirmed by the analysis of CD34+ cells enriched from the two cells sources. However, in vitro stimulation of these progenitor cells using IL3, GM-CSF, erythropoietin and steel factor (SF) revealed that, after 48 h in suspension culture, up to 30% of the CD34+ cells were in S/G2m phase. The fact that cycling CD34+ cells are only detectable in BM but not in PB or CB may suggest different adhesive properties of migrating/mobilized 'stem cells' which may require the BM micro-environment for adequate proliferation in vivo.

Adult↗
PubMed1996

The composition of CD34 subpopulations differs between bone marrow, blood and cord blood.

Our previous data obtained by flow cytometry and by clonogenic assay had consistently shown a lower cloning efficiency of hematopoietic progenitor cells in bone marrow (BM) compared to those in blood (PB) or in cord blood (CB). Also, recent clinical reports have described more rapid reconstitution after PB than after BM transplantation. We have applied two- or three-color flow cytometric analysis using monoclonal antibodies directed against the stem- and progenitor cell antigen CD34, in combination with other cell surface markers. We report significant differences in the composition of progenitor cells contained in BM (238 specimens from 53 healthy donors and from patients in remission), PB (301 samples from 92 patients with or without cytokine support) and CB (n = 37). Leukapheresis products (Pher, n = 69) were included in the study as well as positively selected CD34+ cells obtained from BM (BMsel, n = 2), PB (PBsel, n = 28) and CB (CBsel, n = 5). We used monoclonal antibodies directed against CD7, CD19, CD34, CD38, CD45RA and glycophorin A. The highest proportion of CD34+ cells (in % of the MNC) was found in BM (mean 5.37% +/- 4.5). In the other sources, the mean values were 1.79% +/- 2.46 (PB), 1.48% +/- 1.81 (Pher) and 1.1% +/- 1.69 (CB). However, BM was the only source in which a considerable proportion of the CD34+ cells coexpressed the B cell antigen CD19 (mean 30.1%, median 28, range 0 to 84%). The amount of earlier myeloid progenitors as determined by their non-expression of the CD45RA antigen was lowest among BM CD34+ cells (26.7% +/- 16.6). In the other sources, the respective values were 57.5% +/- 17.9 (PB), 63.6% +/- 13.9 (Pher) and 70.4% +/- 16.1 (CB). These results were confirmed by subtype analyses of the CD34+ cells positively selected from the three sources. Enrichment showed minor CD34+ subpopulations to be identified. The mean proportions of B cell progenitors were 0.11% +/- 0.24 (PBsel) and 1.3% +/- 1.42 (CBsel) of the CD34+ cells. The CD34+ cells from all cell sources coexpressed GPA (median 0.15%, range 0 to 1.8%) and CD7 (median 0.25%, range 0 to 1.2%). The proportion of CD38- cells ranged from 0.7 to 4% of the CD34+ MNC. Thus, despite higher CD34 counts in BM, the relative proportions of myeloid progenitors are higher in PB and in CB. This suggests that, if timely reconstitution depends on the number of CD34+ cells transplanted, the mean number of "stem cells' (SC) required is 1.4-fold (for myeloid cells) or 2.2-fold (for earlier myeloid cells) higher for BM than for PB.

Adolescent↗
PubMed1995

Characterization of hematopoietic stem cells.

On the basis of density-separated mononuclear cells isolated from bone marrow, peripheral blood, and cord blood, we have repeatedly shown good correlation between two-color flow cytometric (FACS) CD34 analysis and colony formation in the clonogenic assay. We have analyzed the distributions of CD34 subpopulations in these three stem cell sources using patients' and donors' bone marrow biopsies (n = 196), cord blood samples from full-term deliveries (n = 14), and peripheral blood from patients mobilized by chemotherapy and/or cytokine treatment (n = 258). Irrespective of absolute cell counts, the mean (+/- SD) proportions of CD34+ cells were clearly higher in bone marrow (5.6 +/- 4.6% of mononuclear cells) than in peripheral blood (1.9 +/- 2.6) and cord blood (1.7 +/- 2.6). However, two-color FACS analyses revealed significant differences among these cell sources with regard to their distribution of CD34 subpopulations: B-cell progenitors coexpressing CD34 and CD19, at considerable concentrations of > 0.5%, were only found in bone marrow (mean 30 +/- 24.3% of CD34+ mononuclear cells, median 28.7%, minimum 0%, maximum 83.3%). In addition, CD34+ cells in S/G2M phase were never detected in peripheral blood or cord blood, but only in bone marrow at a concentration of 10-15% of CD34+ mononuclear cells. On the other hand, the proportions of relatively immature myeloid progenitors, as characterized by not expressing CD45RA and by higher clonogenic capacity, were significantly higher in cord blood (76.7 +/- 17.2) and peripheral blood (58.2 +/- 17.5) than in bone marrow (26.4 +/- 16.7). These data were confirmed by analysis of apheresis products and of progenitors positively selected from different cell sources, and they may explain why, in autologous transplantations of analogous amounts of CD34+ cells, peripheral blood is superior to bone marrow. We conclude from our results that if successful transplantation and timely recovery depend on the number of CD34+ cells transplanted, the mean amount of stem cells required is 1.4- (for myeloid cells) or 2.2-fold (for early myeloid cells) higher for bone marrow than for peripheral blood.

Antibodies, Monoclonal↗
PubMed1994

Flow cytometric assessment of human MIC2 expression in bone marrow, thymus, and peripheral blood.

The cell-surface expression of the MIC2 antigen defined by the monoclonal antibody 12E7 was investigated on human leukocytes in bone marrow (BM), thymus, and peripheral blood (PB) using multiparameter flow cytometry and cell sorting. In contrast to preceding reports, we found that the MIC2 antigen is not restricted to T cells and monocytes. We show that it is also expressed in the B cell and in the granulocytic lineage, the levels of expression being related to distinct maturational stages. CD34+ cells of BM were found to express the antigen at high levels. Along the granulocytic maturation pathway from CD34+CD33+ blasts to mature granulocytes, MIC2 densities appeared progressively reduced with a considerable decline at the myelocyte stage. In B lymphopoiesis, the earliest CD34+ CD10+ B-cell precursor (BCP) cells, further subdivided by expression of CD19, displayed the highest MIC2 density of BM leukocytes. All later BCP stages showed lower MIC2 expression levels, with a remarkable reduction concomitant with loss of the CD34 antigen at the CD10+CD20- surface mu-chain- stage, and a subsequent slight upregulation along with maturation to CD10-CD20high surface mu-chain+ BCPs. The brightest MIC2 expression of all cells tested was displayed by the most immature thymic T-lineage cells characterized by the antigenic profile CD34weakor- CD7++ surface CD3-CD1a(weak) CD4weak CD8-or weak. Common thymocytes stained slightly less intense with 12E7, whereas all subsequent stages of T-lineage cells in thymus, PB, or BM showed markedly reduced MIC2 levels. Mature peripheral CD4+ as well as CD8+ T cells displayed a bimodal distribution of MIC2. In the CD4+ population, the distinct MIC2 levels were related to the well-studied functional subdivision by differential expression of CD45 isoforms, the helper-inducer/memory subset showing higher MIC2 expression than helper-suppressor/naive CD4+ T cells. Similarly high MIC2 densities were found on CD16+ natural killer cells and on CD14+ monocytes, whereas mature peripheral B cells exhibited low or intermediate expression, and granulocytes exhibited no or only dim expression. These results document that the MIC2 antigen (1) is expressed on all leukocyte lineages; (2) is differentially expressed during T- and B-lymphoid, as well as granulocytic maturation; (3) shows highest expression in the most immature lymphocytic and granulocytic developmental stages; and (4) is also differentially expressed on functional T-cell subsets. We speculate that these observations imply a functional significance of MIC2 in the network of hematopoietic adhesion pathways.

12E7 Antigen↗
PubMed1994

Does cord blood contain enough progenitor cells for transplantation?

We analyzed 125 blood samples obtained from umbilical cord immediately after delivery of full-term neonates. Between 0.1 and 10.4% (mean 1.13%, SD 1.34) of the density-separated glycophorin A (GPA)-negative mononuclear cells (MNC) expressed CD34 as analyzed by flow cytometry. These hematopoietic progenitor cells did not coexpress CD19, and the majority were negative for CD45RA. The number of MNC determined per ml cord blood ranged from 1 x 10(5) to 200 x 10(5) (mean 20.2 x 10(5), SD 24.7). Regression analysis revealed that a mean of 56% (n = 26, R = 0.8) and 120% (n = 35, R = 0.94) of the analyzed CD34+ MNC gave rise to day 14 colonies in the clonogenic assay when cultured without or with stem cell factor (SCF). The number and the exact phenotype of progenitor cells required for successful transplantation are not known. If the transplantation of 5 x 10(5) CD34% cells/kg body weight is required for engraftment and one-third of the progenitor cells are lost to cell processing, and if 180 ml blood can be collected from a single umbilical cord (and placenta), our data suggest that 90% of the collections do not contain enough precursors to transplant a 25 kg recipient. To meet these conditions, an average of 1439 ml cord blood would be necessary for transplantation.

Colony-Forming Units Assay↗