Method for determining the haemoglobin content of an erythroid cell
AssigneesUniversite Paris Est Creteil Val De Marne, Assistance Publique-Hopitaux de Paris, Etablissement Franais Du Sang, Institut National de la Sante et de la Recherche Medicale
InventorsMarie Cambot, Gaetana Vandemeulebrouck, France Noizat Pirenne, Pablo Bartolucci, Marie Georgine Rakotoson, Frédéric Galacteros, Nicolas Hebert
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Abstract
The invention concerns a method for determining, by flow cytometry, the hemoglobin content of each erythroid cell of a set of erythroid cells. This method applies in particular to determining the hemoglobin content of each red blood cell of a set of red blood cells. The invention also concerns a method for determining the amount of red blood cells transfused into a patient and for monitoring the therapeutic efficacy of a treatment for sickle cell disease or β-thalassemia.
Claims20
A method for determining, in vitro, a content of at least one hemoglobin Hbx in each erythroid cell of a set of erythroid cells contained in a sample of erythroid cells, comprising the steps of:
- a) establishing a standard curve associating fluorescence intensity measured for erythroid cells with at least one hemoglobin Hbx content, using a homogeneous sample of erythroid cells:
- b) isolating erythroid cells from the sample;
- c) permeabilizing a membrane of the isolated erythroid cells;
- d) labeling the at least one hemoglobin Hbx of the erythroid cells obtained in step b) with at least one anti-Hbx antibody conjugated to a fluorochrome capable of emitting a fluorescence;
- e) measuring, by flow cytometry, fluorescence intensity (MFI) of each erythroid cell of the set of erythroid cells;
- and
- f) determining the content of the at least one hemoglobin Hbx in each erythroid cell of the set of erythroid cells by comparing the fluorescence intensity of each erythroid cell with the standard curve established in a),
- wherein establishing the standard curve comprises:
- establishing a calibration straight line which makes it possible to correlate a fluorescence intensity with a fluorophore number;
- obtaining a calibration blood sample from patients having an Hbx content that is homogeneous over all of their erythroid cells and having the Hbx labeled with an anti-Hbx antibody conjugated to a fluorophore;
- relating a measurement of fluorescence intensity in each erythroid cell in the calibration blood sample to the calibration straight line, such that it is possible to deduce an amount of fluorophore in each erythroid cell;
- deducing a number of Hbx molecules of each erythroid cell from the amount of fluorophore in each erythroid cell;
- determining a mean content of the at least one hemoglobin Hbx per erythroid cell (MCHbxCo) from the number of Hbx molecules of each erythroid cell;
- and
- establishing the standard curve by associating the MCHbxCo and a measurement of the fluorescence intensity in each erythroid cell in said calibration blood sample.
Dependencies are inferred from the claim text.