Detection of chromosomal aneuploidies in fetal cells isolated from maternal blood using single-chromosome dual-probe FISH analysis.

Detection of chromosomal aneuploidies using fetal cells isolated from maternal blood, for prenatal non-invasive genetic investigation, has been a long-sought goal of clinical genetics to replace amniocentesis and chorionic villous sampling to avoid any risk to the fetus. The purpose of this study was to develop a sensitive and specific new assay for diagnosing aneuploidy with circulating fetal cells isolated from maternal blood as previously reported using two novel approaches: (i) simultaneous immunocytochemistry (ICC) evaluation using a monoclonal antibody for i-antigen, followed by fluorescence in situ hybridization (FISH); (ii) dual-probe FISH analysis of interphase nuclei using two differently labeled probes, specific for different loci of chromosomes 21 and 18; in addition, short tandem repeats (STR) analysis on single cells isolated by micromanipulation was applied to confirm the presence of fetal cells in the cell sample enriched from maternal blood. Blood samples were obtained from women carrying trisomic fetuses, and from non-pregnant women and men as controls. Using ICC-FISH approach, a large heterogeneity in immunostaining pattern was observed, which is a source of very subjective signal interpretation. Differently, dual-probe FISH analysis provided for a correct diagnosis of all pregnancies: the mean percentage of trisomic cells was 0.5% (range, 0.36-0.76%), while the mean percentage of trisomic cells in the control group (normal pregnancies or non-pregnant women) was ≤0.20%. The application of the dual-probe FISH protocol on fetal cells isolated from maternal blood enables accurate molecular detection of fetal aneuploidy, thus providing a foundation for development of non-invasive prenatal diagnostic testing.

Hemopoiesis in the liver of adult tumor-bearing mice.

In the liver of adult mice bearing an Ehrlich carcinoma on the leg, progressively hypoxic and displaying reactive hepatitis but not metastatic dissemination, extramedullary hemopoiesis was detected. Electron microscopy revealed mainly erythropoietic islands and scattered megakaryocytes in maturation stages up to the platelet-releasing phase. Erythropoietic cells expressed an embryonic-type of hemoglobin, which is more adequate to oxygenate hypoxic environments than the adult type. They were positive for the peroxidase reaction due to the presence of hemoglobin and could furthermore be visualized by the blue-excited red autofluorescence of protoporphyrin IX. Extramedullary hemopoiesis, one of the various examples of reactivation of fetal features in the liver associated with carcinogenesis, is supposed to be compensatory for the loss of blood cells induced by the tumor. Reviewing this process has the purpose of raising the question whether the fetal features are better adapted than adult ones to the metabolic and physiological characteristics of a tumor-influenced organism.

Fetal erythroblast isolation up to purity from cord blood and their culture in vitro.

BACKGROUND: Erythroblasts have been the most encouraging candidate cell type for noninvasive prenatal genetic investigation. We previously showed that human erythroblasts can be recovered from bone marrow and blood bank buffy coats by a physical cell separation. In the present study, we modified our previous methodology, taking into account the peculiar behavior of erythroblasts in response to modifications of pH and osmolality of the separation medium. METHODS: Twenty to forty milliters of cord blood were initially centrifuged on Ficoll/diatrizoate (1.085 g/ml). The interphase cells were further separated on a continuous density gradient (1.040-1.085 g/ml). Two different gradients were initially compared: the first was iso-osmolar and neutral, whereas the second also contained an ionic strength gradient and a pH gradient (triple gradient). A subsequent monocyte depletion was performed by using magnetic microbeads coated with anti-CD14 monoclonal antibody (mAb), and erythroblasts were purified by sedimentation velocity. Purified cells were investigated by analyses with fluorescence-activated cell sorting (FACS) and fluorescence in situ hybridization (FISH) and immunocytochemistry with mAb against fetal hemoglobin and were cultured in vitro. RESULTS: When nucleated cells were spun on an iso-osmolar and neutral continuous density gradient, two separated bands of nucleated red blood cells (NRBCs) were obtained: a light fraction banding at 1.062 g/ml and an heavy fraction banding at 1.078 g/ml. Conversely, when cells were spun in the triple gradient, NRBCs were shifted to the low-density region. Monocyte depletion by immunomagnetic microbeads and velocity sedimentation provided a pure erythroblast population. FACS and FISH analyses and immunocytochemistry substantiated the purity of the isolated cell fraction, which was successfully cultured in vitro. CONCLUSIONS: We have shown that fetal erythroblasts can be purified up to homogeneity from cord blood, but further refinements of the isolation procedure are necessary before the same results can be obtained from maternal peripheral blood.

Characterization of the biophysical properties of human erythroblasts as a preliminary step to the isolation of fetal erythroblasts from maternal peripheral blood for non invasive prenatal genetic investigation.

BACKGROUND AND OBJECTIVE: Fetal erythroblasts in maternal circulation represent a valuable source of fetal cell material which can be obtained with non-invasive procedures that do not endanger the fetus. Physical separation techniques have been invaluable in the isolation and characterization of different cells. There are basically two principles that have been used most successfully: separation according to density and separation according to size. In order to determine whether physical separation procedures are capable of purifying human erythroblasts, the biophysical characteristics of these cells were determined. METHODS: Bone marrow particles were obtained from formal adults and peripheral blood buffy coats from blood banks. A single cell suspension was initially fractionated by buoyant density gradient centrifugation. Fractions enriched in erythroblasts were pooled and further processed by velocity sedimentation in order to take advantage of the differences in size of erythroblasts and other cells. RESULTS: Density distribution curves were drawn after density gradient centrifugation for the different cell types present in the starting cell samples. Separation of the erythroblast-enriched density fractions by velocity sedimentation was successful and a highly purified population of erythroblasts was obtained. Cell size distribution of the different cell types was determined. INTERPRETATION AND CONCLUSIONS: This initial study defines the biophysical properties (size and density) of human erythroblasts in bone marrow and peripheral blood and is a necessary preliminary step in setting up the optimal procedure for the isolation of fetal erythroblasts from maternal peripheral blood in sufficient amounts and purity for prenatal non-invasive genetic investigation.

Giant cell formation in Hodgkin's disease.

The identity of Reed-Sternberg cells in Hodgkin's disease has remained an unresolved issue, though many studies have addressed this question. Giant cells are usually formed either by endomitosis without cytoplasmic division or by cell fusion through cytokines or viruses. Growing evidence associates Epstein-Barr virus (EBV) with Hodgkin's disease, a major issue being whether EBV is a passenger virus or has an aetiological role. This communication describes experimental conditions enabling observation of giant cell cytogenesis from peripheral blood mononuclear cells in culture. Mononuclear cells were isolated from autologous peripheral blood and cocultured with a single-cell suspension obtained from Hodgkin's lymph nodes in a culture chamber where the two cell populations are isolated by a microporous membrane that allows only cytokines and viruses to pass through. Under these experimental conditions, giant cells are formed in the peripheral blood mononuclear cell fraction; some of them appear morphologically indistinguishable from Reed-Sternberg cells and their mononuclear variant, while others much resemble Langhans giant cells. Some of these giant cells are positive for EBV DNA by in situ hybridization. These results suggest that an EBV-dependent biological activity is responsible for giant cell cytogenesis originating from lymphocytes and monocytes, induced either by EBV and/or cytokines.

Role of H2-receptor antagonists in the treatment of duodenitis.

Non-erosive duodenitis is an ulcer-independent disorder, the pathogenesis of which is still unclear but apparently unrelated to gastric hyperacidity. However, antisecretory agents such as H2-blockers can be effective not only in relieving dyspeptic symptoms but also in promoting endoscopic healing or improvement. In this respect conflicting data are reported with cimetidine while promising, although preliminary, results were obtained with ranitidine and with nizatidine. Nizatidine seems especially effective when administered at a dose of 150 mg b.i.d., a regimen providing moderate, but continuous acid inhibition throughout a 24 hour period.

Isolation of Reed-Sternberg cells from lymph nodes of Hodgkin's disease patients.

This study describes a simple and relatively rapid method of purifying Reed-Sternberg (R-S) cells and their morphologic variants from the lymph nodes of patients affected by Hodgkin's disease. Our initial studies defined the optimal procedure for a quantitative disaggregation of Hodgkin's lymph nodes and the densities of R-S cells in several donors. These preliminary steps were helpful in the development of strategies for selectively concentrating R-S cells by density gradient centrifugation. We layered a single-cell suspension over Percoll of appropriate density, centrifuged these samples for 15 minutes, and collected a fraction enriched in R-S cells. Most of the R-S cells were distributed between densities of 1.060 and 1.072, with a peak at approximately 1.066 g/mL. R-S cells are denser than many mononuclear cells present in the lymph nodes of Hodgkin's patients and lighter than reactive cells such as eosinophils, mast cells, and neutrophils. However, the ranges of densities of these cell types overlap, making purification of R-S cells by isopyknic centrifugation impossible. Nevertheless, when this enriched fraction is further processed by velocity sedimentation in order to take advantage of the larger size of R-S cells as compared with all other cells, a substantial purification is achieved. We used three different velocity-sedimentation chambers to find the optimal conditions for obtaining the highest purity with a high final yield. The cells isolated by this method are viable, appear to be morphologically normal, and have been further characterized biologically.

Physical procedures for the separation of blood and marrow cells.

Most of the questions being asked in contemporary experimental hematology research can be framed in terms of the properties of the individual cells in the system and the interactions among those cells. Physical separation procedures are among the most powerful methods for the purification of homogeneous cell populations, and during the past decades, thousands of reports have described the separation of many different cells using these techniques. Theoretical and technical improvements have been rapidly transferred from the research laboratory into the construction of blood cell separation machines, which have proved of exceptional support in the practice of clinical hematology.

Isolation of human monocytes according to volume and density.

Human peripheral blood monocytes were obtained in very pure preparations by using buoyant density gradient centrifugation followed by velocity sedimentation. We introduced several modifications in the experimental procedure in order to take advantage of the minimal differences in cell density and size between monocytes and lymphocytes. Previous methods used to isolate monocytes based on their physical properties are reviewed, and the theory of velocity sedimentation is discussed with regard to the differences in the methodology used, which account for the different results we obtained.

Isolation of normal human megakaryocytes.

This paper reports a simple procedure for obtaining human megakaryocytes with a high purification and high recovery yield. Bone marrow cells, obtained from surgically removed ribs, were separated by a two-step procedure. Initially, a single cell suspension was enriched in megakaryocytes by equilibrium density centrifugation, the low density cell fraction was subsequently layered over a shallow continuous albumin gradient in a glass sedimentation chamber. Megakaryocytes averaged 0.03 +/- 0.02% of all nucleated cells in the starting marrow cell suspension, after this procedure an average 80 +/- 15% of the initial megakaryocyte population was recovered with a purity of 94 +/- 4%. Previous methods, based upon the use of a two-step procedure, are reviewed. The theory of velocity sedimentation is discussed with regard to the differences in the methodology used, which account for the different results I obtained.

The ultrastructural localization of factor VIII-antigen in human platelets, megakaryocytes and endothelial cells utilizing a ferritin-labelled antibody.

By means of electron microscopy combined with the use of monospecific anti-factor VIII-antigen (VIIIR:AG) antibodies conjugated to ferritin, the subcellular localization of VIIR:AG in platelets, megakaryocytes and in endothelial cells has been established. The reported results suggest the possibility that the megakaryocyte is able to synthesize VIIIR:AG and also to secrete it by means of a microcanalicular system similar to that present in the endothelial cell. Platelets may derive their VIIIR:AG content partly from the megakaryocyte and partly from the plasma.

Problems in the detection of carriers of haemophilia A.

Factor VIII activity and factor VIII related--or Willebrand--antigen were studied in 49 known carriers of haemophilia A and 31 normal women, and the data were analysed by four statistical approaches. Sixteen per cent of normals and 18% of carriers were misclassified, overlapping with the other group. Although the percentage of carriers detected is higher when taking into account the results of both biological and immunological factor VIII, it is lower than others recently reported, and the discrepancies between the results obtained are discussed.