US 12,345,718 B2
Method for determining the haemoglobin content of an erythroid cell
Marie Cambot, L'Haÿ-les-Roses (FR); Gaetana Vandemeulebrouck, París (FR); France Noizat Pirenne, Paris (FR); Pablo Bartolucci, L'Haÿ-les-Roses (FR); Marie Georgine Rakotoson, Antananarivo (MG); Frédéric Galacteros, Santeny (FR); and Nicolas Hebert, Ollainville (FR)
Assigned to UNIVERSITÉ PARIS EST CRÉTEIL VAL DE MARNE, Creteil (FR); ASSISTANCE PUBLIQUE - HÔPITAUX DE PARIS, Paris (FR); ÉTABLISSEMENT FRANAIS DU SANG, La Plaine Saint Denis (FR); and INSTITUT NATIONAL DE LA SANTÉ ET DE LA RECHERCHE MÉDICALE, Paris (FR)
Filed by UNIVERSITÉ PARIS EST CRÉTEIL VAL DE MARNE, Créteil (FR); Assistance Publique—Hôpitaux de Paris, Paris (FR); ÉTABLISSEMENT FRANÇAIS DU SANG, Saint-Denis (FR); and Institut National de la Santé et de la Recherche Médicale, Paris (FR)
Filed on Aug. 6, 2021, as Appl. No. 17/395,969.
Application 17/395,969 is a continuation in part of application No. 16/347,738, granted, now 11,231,427, previously published as PCT/FR2017/053015, filed on Nov. 3, 2017.
Claims priority of application No. 1660713 (FR), filed on Nov. 4, 2016.
Prior Publication US 2022/0011324 A1, Jan. 13, 2022
Int. Cl. G01N 33/72 (2006.01); G01N 21/64 (2006.01); G01N 33/49 (2006.01)
CPC G01N 33/721 (2013.01) [G01N 33/723 (2013.01); G01N 2333/805 (2013.01); G01N 2800/22 (2013.01); G01N 2800/52 (2013.01)] 20 Claims
 
1. 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.