PubMed · 4167282
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1967-10-14. The E.S.R.. https://pubmed.ncbi.nlm.nih.gov/4167282/
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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.
One of the most common health criteria--erythrocyte sedimentation rate (ESR)--is considered in the paper. It is shown that the simple model presented, based on the generalized Stokes formula, the blood volume conservation law, and the Smoluchowski theory of particles coagulation, makes it possible, on the basis of experimentally recorded sedimentation curves, to identify quantitatively the values of the essential physical parameters of the coupled processes of erythrocyte aggregation and sedimentation. The analytical solution of Smoluchowski equation is used to evaluate the sedimentation and aggregation rate constants. The problem of determining the erythrocyte aggregation rate (EAR) is transformed to a minimization task in which only the experimental results for ESR are needed. Experimentally ESR is measured accurately enough by using an equipment set up just for the purpose. This method of identification could be used as a diagnostic test in hematological laboratories.
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