Method for determining the haemoglobin content of an erythroid cell

Patent metadata

Field	Value
Publication number	US-2022011324-A1
Application number	US-202117395969-A
Country	US
Kind code	A1
Filing date	Aug 6, 2021
Priority date	Nov 4, 2016
Publication date	Jan 13, 2022
Grant date	—

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.

First claim

1 . A method for determining, in vitro, the 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) isolating erythroid cells from the sample; b) permeabilizing the membrane of the isolated erythroid cells; c) 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; d) measuring, by flow cytometry, the fluorescence intensity (MFI) of each erythroid cell of the set of erythroid cells; e) 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 red blood cell with a standard curve associating the fluorescence intensity measured for a red blood cell with the at least one hemoglobin Hbx content.

2 . The method as claimed in claim 1 , wherein the membrane of the isolated erythroid cells is fixed before the permeabilization step.

3 . The method as claimed in claim 1 , wherein said sample is a blood sample.

4 . The method as claimed in claim 1 , wherein said fluorochrome is selected from the group consisting of phycoerythrin (PE), fluorescein, isothiocyanate, a derivative thereof or a combination thereof.

5 . The method as claimed in claim 1 , wherein the at least one Hbx hemoglobin is at least one first hemoglobin Hbx1, at least one second hemoglobin Hbx2 and at least one n n th hemoglobin designated as Hbxn, said method comprising the steps of:
a) isolating erythroid cells from a sample; b) permeabilizing the membrane of the isolated erythroid cells; c 1 ) labeling the at least one first hemoglobin Hbx1 of the erythroid cells obtained in step b) with at least one anti-Hbx1 antibody conjugated to a first fluorochrome capable of emitting a first fluorescence; labeling the at least one second hemoglobin Hbx2 of the erythroid cells obtained in step b) with at least one second anti-Hbx2 antibody conjugated to a second fluorochrome capable of emitting a second fluorescence; labeling the at least one n th hemoglobin Hbxn of the erythroid cells obtained in step b) with at least one anti-Hbxn antibody conjugated to a n th fluorochrome capable of emitting a n th fluorescence, d 1 ) measuring, by flow cytometry, the fluorescence intensity of each fluorescence emitted by the first, the second, the n th fluorochrome of each erythroid cell of a set of erythroid cells; e 1 ) determining the content of the at least one first hemoglobin Hbx1, the at least one second hemoglobin Hbx2, the at least one n th hemoglobin Hbxn in each erythroid cell of the set of erythroid cells by comparing each of the first, the second, the n th fluorescence intensities measured in step d 1 ) with a first, a second and an n th standard curve associating the measured first, second, and n th fluorescence intensities for a red blood cell, with content of the at least one first hemoglobin Hbx1, the content of the at least one second hemoglobin Hb2 and the content of the n th hemoglobin Hbxn.

6 . The method as claimed in claim 1 , wherein the at least one Hbx hemoglobin are n hemoglobins Hbx, designated as Hbxn, said method comprising the steps of:
a) isolating erythroid cells from a sample; b) permeabilizing the membrane of the isolated erythroid cells;
c 1 ) labeling the at least one first hemoglobin Hbx1 of the erythroid cells obtained in step b) with at least one anti-Hbx1 antibody conjugated to a fluorochrome;
d 1 ) measuring, by flow cytometry, the fluorescence intensity (MFI) of each erythroid cell of a set of erythroid cells;
e 1 ) determining the content of the at least one first hemoglobin Hbx1 in each erythroid cell of the set of erythroid cells by comparing the fluorescence intensity of each red blood cell with a standard curve associating the fluorescence intensity measured for a red blood cell with the first hemoglobin Hbx1 content;
f) iterating the set of steps c 1 )-e 1 ) for each of the at least one second Hbx2 (steps c 2 )-e 2 )), at least one third Hbx3 (steps c 3 )-e 3 )) and n th Hbxn (steps c n )-e n )) until the content of the n hemoglobins Hbx in each erythroid cell of the set of erythroid cells is determined.

7 . The method as claimed in claim 1 , wherein said at least one anti-Hbx antibody is directed against at least one of the chains of the at least one hemoglobin Hbx that are selected from the group consisting of α, β, γ, δ, ε (chain, glycosylated derivatives thereof, blood disease variants thereof, mutated forms thereof, or a mixture thereof.

8 . The method as claimed in claim 1 , wherein said at least one hemoglobin Hbx is selected from the group consisting of HbF, HbA, HbS and a combination thereof.

9 . The method as claimed in claim 1 , wherein the membrane of the isolated erythroid cells is permeabilized with sodium dodecyl sulfate.

10 . The method as claimed in claim 2 , wherein the membrane of the isolated erythroid cells is fixed with sodium azide and/or formaldehyde.

11 . The method as claimed in claim 1 , wherein the content of the at least one hemoglobin Hbx is determined for each erythroid cell of a set of at least 10,000 erythroid cells.

12 . The method as claimed in claim 1 , wherein the erythroid cells are red blood cells.

13 . The method as claimed in claim 1 , wherein the content the at least one hemoglobin Hbx in each erythroid cell of the set of erythroid cells is expressed as a concentration relative to the volume of the erythroid cells.

14 . A method for determining, in vitro, an amount of red blood cells transfused into a patient suffering from sickle cell disease, alpha-thalassemia or beta-thalassemia, comprising:
a) determining the content of the at least one hemoglobin Hbx selected from the group consisting of HbA, HbF and HbS of each red blood cell of a set of red blood cells of a sample of red blood cells from the patient according to the method of claim 12 ; b) using the results of step a) to determine the amount of red blood cells transfused into a patient, said transfused red blood cells having an HbF and/or HbS content substantially equal to zero (=0 pg) and or a content ratio of the HbS/(HbF+HbA) substantially equal to zero (=0).

15 . An in vitro method for monitoring the therapeutic efficacy of a Hematopoietic stem cell transplantation (HSCT) or of a treatment for myelodysplastic syndromes, sickle cell disease or for β-thalassemia, comprising:
a) obtaining a sample containing red blood cells from a patient having undergone a HSCT or a treatment for a myelodysplastic syndrome, sickle cell disease or for β-thalassemia;
b) determining the content of the at least one hemoglobin Hbx in each red blood cell of a set of red blood cells of said sample according to the method of claim 12 ;
c) using the results of step b) in the monitoring of the therapeutic efficacy of Hematopoietic stem cell transplantation (HSCT) or the treatment for sickle cell disease or for β-thalassemia, in which a therapeutic efficacy is observed when at least a predetermined percentage of the red blood cells of the set of red blood cells has a content of the at least one hemoglobin Hbx that increases or decreases at least 2%, at least 5%, at least 7%, at least 10%, at least 12%, at least 15%, or at least 20% compared to the same content prior to the HSCT or the treatment for myelodysplastic syndromes, sickle cell disease or for β-thalassemia.

16 . A method for treating sickle cell disease or β-thalassemia, comprising:
a) obtaining a sample contai

Assignees

• Univ Paris Val De Marne
• Hopitaux Paris Assist Publique
• Francais Du Sang Ets
• Inst Nat Sante Rech Med

Inventors

• Cambot Marie
• Vandemeulebrouck Gaetana
• Noizat Pirenne France
• Bartolucci Pablo
• Rakotoson Marie Georgine
• Galacteros Frédéric
• Hebert Nicolas

Classifications

• G01N33/72 Primary

  involving blood pigments, e.g. haemoglobin, bilirubin {or other porphyrins; involving occult blood} · CPC title

• A61P7/00

  Drugs for disorders of the blood or the extracellular fluid · CPC title

• G01N33/80

  involving blood groups or blood types {or red blood cells (white blood cells G01N33/56972)} · CPC title

• G01N2800/52

  Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis · CPC title

• G01N33/721 Primary

  Haemoglobin · CPC title