FIELD OF THE INVENTION:

The invention relates to a method for in vitro determination of the relative age evaluation of subpopulation of red blood cells by using intracellular glycated hemoglobin measurement by flow cytometry.

The invention also relates to a novel method for in vitro diagnostic of hemolytic anemia in a patient, and to a method for monitoring the therapeutic efficacy of a treatment of red blood cell-related disorder by using intracellular glycated hemoglobin measurement by flow cytometry.

BACKGROUND OF THE INVENTION:

Chronic or acute hemolysis is defined as an imbalance between the rate of red blood cell (RBC) production and destruction. Severity of hemolysis is mainly appreciated through the total hemoglobin (Hb) concentration in blood measurement. Consequently, depending on the degree of importance of the imbalance, hemolysis results in low, moderate or severe anemia in patients. Whatever the cause, hemolysis results in an overall reduced lifespan of RBCs, as they are prematurely removed from the circulation.

Lifespan of normal RBCs has been widely studied and established to be 110-120 days. It can be assessed by a wide variety of in vivo and in vitro methods. Reference in vivo techniques are based on the use of a label that is ex vivo linked to RBCs allowing their detection over time after reinjection while they age in the circulation. In these methods, label is either radioactive isotope such as ⁵¹ Cr or non-radioactive biological molecule such as biotin. Alternative consists in orally administrated ¹⁵ N-glycine that is then incorporated in RBCs, allowing their detection. Recently, a carbon monoxide (CO) production method has been described. This method measures CO molecules released by the heme degradation to assess RBCs turnover and lifespan. Given some difficulties of in vivo methods that render them avoided in clinical practice, investigators have attempted to use in vitro methods for age correlative separation of red cells. The most commonly used method based on this approach is that of density dependent cell separation. Indeed, the investigation with radioactive ⁵⁹ Fe labeled normal RBC revealed that as red cells age in circulation, they slowly dehydrate, shrink, and become smaller and denser over the entire life. Therefore, bottom of layer of centrifuged cells (High-density) was enriched with old RBCs, contrary to young RBCs that were concentrated in the top layer of centrifuged cells (low-density) [1], As they age, normal RBCs exhibit physiological changes, such as dehydration, a decrease in activities of the intracellular enzymes and an increase in Hb concentration. Glycated Hb (the most abundant being HbAlc form) also increases, as RBC age. The glycation of Hb is a non-enzymatical reaction that is affected by the plasmatic concentration of glucose and the time RBCs last in the circulation. In non-diabetic subjects, the Hb glycation only depends on the RBC lifespan. In addition, it was demonstrated that the rate of glycation according to the lifespan is linear [2], To date, current methods to assess RBC lifespan are invasive and time consuming, requiring infusion of autologous labeled RBCs and long follow-up.

The most common hemolytic anemia syndrome is a major symptom of sickle cell disease (SCD), an autosomal recessive blood disorder caused by a point mutation in the HBB gene encoding the P-globin subunit. It leads to the substitution of the hydrophilic glutamic acid at the sixth position by a hydrophobic valine, generating sickle hemoglobin (HbS, a2p ^s 2) [3], SCD is characterized by a complex pathophysiology and by multi-systemic consequences that are driven by the polymerization of HbS [4], The latter occurs under deoxygenated condition and is extremely sensitive to its concentration inside red blood cells (RBCs). HbS-induced changes in RBC biological and rheological properties result in obstruction of various microcirculatory vessels, leading to vaso-occlusive events, as well as hemolytic anemia.

RBCs survival from individuals with SCD is reduced to one-sixth of normal RBC (15- 20 days) [5], Sickle RBC lifespan is controlled by many determinants, its most powerful modulator being fetal hemoglobin (HbF, a2y2) [6], Fetal Hb (HbF) is the main clinical modulator of SCD severity by inhibiting HbS polymerization, a protective effect that relies on its quantity and its distribution among the RBC population. It is both a result of reduction in mean cell HbS concentration and a direct inhibition of HbS polymerization because neither HbF nor its mixed hybrid tetramer (a2p ^s y) can enter the deoxy -HbS polymer phase [7], HbF- induced improved sickle RBC survival was confirmed in a study in which they showed that F- cells (the subset of cells in which HbF can be detected) survived much longer than sickle RBCs with no detectable HbF (non F-cells) [8],

To investigate the beneficial effect of HbF on the sickle RBC lifespan prolongation, all the previously mentioned methods are not suitable. Besides they are labor-intensive, cumbersome, and invasive methods, the most of in vivo approaches can only measure the mean RBC lifespan not taking into account the distribution. However, HbF is often heterogeneously distributed in RBC from SCD patients and therefore, is not a good predictor of all circulating sickle RBC behavior or SCD clinical severity [9-10], In the case of in vitro methods, the same question is raised about the applicability of the density-dependent separation technique. Actually, density cannot be used as a surrogate for age in SCD because sickle RBCs show a broad and heterogeneous density distribution. Previous study demonstrated that a subset of young sickle RBCs, including reticulocytes and even the less mature cells displaying transferrin receptor-positive sickle reticulocytes, undergo abnormal in vivo cation loss and dehydration and accelerated increase in cell density due to severe membrane abnormalities when compared with cells with normal hydration [11],

Herein the inventors develop of a new approach that allows the relative age evaluation of individual red cells in different RBC subpopulations using intracellular glycated hemoglobin measurement by flow cytometry. This new approach could allow to diagnostic hemolytic anemia, monitor the efficacy of treatment of red blood cell-related disorder.

SUMMARY OF THE INVENTION:

Glycated Hb was measured and compared between five healthy donors (AA), seven non-diabetic and non -transfused homozygous SCD patients (SS) and five non-diabetic, transfused SS patients. The single cell glycation was assessed by flow cytometry using mouse anti -human HbAlc antibody. The inventors demonstrated that glycated Hb was significantly lower in SS patients than in healthy donors (glycated HbS =3.7%, glycated HbA =5.3%, p=0.0025 by DCA and HPLC respectively, and by flow cytometry p=0.0061). In transfused SS patients, results showed that glycated Hb was lower in autologous SS-RBCs than in AA-RBCs by a factor of four (P= 0.008), thus confirming the characterized reduced lifespan of SS RBCs. Glycated Hb levels were measured in RBC sub-populations based on their HbF level (high HbF or low HbF subfractions) in non-transfused SS patients. Glycated Hb level was lower in low HbF RBCs, as compared to high HbF RBCs (P=0.0023). This difference was not detected in healthy donors, thereby indicating that HbF has an effect on relative age only in SCD patients..

Single cell glycated Hb assessed by flow cytometry allows to simultaneously estimate the relative age of different RBC sub-populations. This new tool is noninvasive and requires only one single time point measurement.

In particular, the present invention is defined by the claims. DETAILED DESCRIPTION OF THE INVENTION:

Definitions

As used herein, the term "set of red blood cells" corresponds to a set of red blood cells contained in a sample of red blood cells. The set of red blood cells may range up to 100% of the red blood cells present in the sample, ie all of the red blood cells present in the sample. Thus, the set of red blood cells corresponds to between 100% and 0%, 0% being excluded, red blood cells present in the sample. In a particular embodiment, the set of red blood cells comprises at least 10,000 red blood cells, preferably at least 100,000 red blood cells, preferably at least 200,000 red blood cells, preferably between 50,000 and 200,000 red blood cells.

As used herein, the term “red blood cell” or “red blood cells”, also called as red cells, red blood corpuscles or erythroid cells, refer to circulating cells that contain hemoglobin for which it is desired to measure the presence of glycated hemoglobin among a set of said cells. Red blood cells includes immature RBC and mature RBC. Reticulocyte, as defined as enucleated, RNA-containing erythroid cell, refers to immature red blood cells produced in the bone marrow and released into the peripheral blood where they mature into mature RBCs (also known as erythrocyte) within 1 to 2 days. Red blood cells are the most common type of blood cell and the vertebrate's principal means of delivering oxygen to the body tissues via blood flow through the circulatory system. RBCs take up oxygen in the lungs and release it into tissues while squeezing through the body's capillaries. The cytoplasm of RBC is rich in hemoglobin. In order to avoid any ambiguity, throughout the present description, the terms "erythroid cell” and "erythroid cells" may be respectively substituted, for example, by red blood cell or red blood cells.

As used herein, the term “hemoglobin” has its general meaning in the art and refers to an iron-containing biomolecule that can bind oxygen and is responsible for the red color of the cells and the blood. Human hemoglobins are composed of 4 subunits of identical polypeptide chains. The subunits vary according to the type of hemoglobin and we can distinguish 4 normal hemoglobins in humans:

- embryonic hemoglobins: hemoglobin Gower I (^2s2), hemoglobin Portland (^2y2), and hemoglobin Gower II (a2s2) ;

- Hemoglobin Fetal (HbF) (a2y2);

- adult hemoglobin (HbA) (a2p2);

- hemoglobin A2 (HbA2) (a262). "HbA" denotes Hemoglobin A, also known as adult hemoglobin, hemoglobinAl or A2. HbA is the most common human hemoglobin tetramer in healthy subjects, accounting for over 97% of the total red blood cell hemoglobin. Hemoglobin A is the most common adult form of hemoglobin and exists as a tetramer containing two alpha subunits and two beta subunits (a2~2). According to a specific embodiment, "level of HbA" denotes the level of HbA and/or HbA2.

"HbF" denotes hemoglobin F composed of two subunits of a (alpha) chains and two subunits of non-alpha (beta, delta or gamma) chains.. HbF is composed of two types of y chains: Gy and Ay which differ in terms of their residue in position 136 corresponding to a glycine for the Gy chain and an alanine for Ay. There are two minor forms of HbF, including HbFl which represents 10% of total hemoglobin in the foetus. Other forms of HbF can be observed but in very small amount.

Numerous Hemoglobin variants have been reported to be associated with a disease, notably a blood disease. Hemoglobin variants includes Hemoglobin Asharon, Hemoglobin Barts (Hb Barts) (y4), Hemoglobin C (HbC) (a2~c2), Hemoglobin D-Punjab (HbD) (a2~D2), Hemoglobin E (HbE) (a2~E2), Hemoglobin H (~4), Hemoglobin Hopkins-2, Hemoglobin S (a2~s2), Hemoglobin AS, Hemoglobin SC disease, Hemoglobin G-Makassar. Hemoglobin variant and mutation that cause hemoglobin-related disorder are well known in the art and are disclosed in Giardine et al, Nucleic Acids Res. 2014 Jan;42; and on https://globin.bx.psu.edu/hbvar/menu.html.

Hemoglobin can be glycated via a non-enzymatical reaction (Amadori reaction) that is affected by the plasmatic concentration of glucose and the time RBCs last in the circulation. Amadori compounds are produced from reducing sugars, such as glucose, and an amine.

As used herein, the term “glycated hemoglobin” also known as “GHb" is a form of hemoglobin that results from a non-enzymatical reaction (i.e Amadori reaction) covalent addition of a sugar molecule to the hemoglobin. Glycated hemoglobin includes several forms of glycation: HbAla corresponds to the link of a fructose 1,6 diphosphate (HbAlal) or a glucose 6 phosphate (HbAla2); HbAlb corresponds to the link of a pyruvate; and HbAlc, the most abundant glycated Hb found in RBCs, contributing for more than 80% of glycated hemoglobin, corresponds to the link of a glucose and is affected by the plasmatic concentration of glucose and the time RBCs last in the circulation. In particular embodiment, the glycated hemoglobin is HbAlc.

As used herein, the term "flow cytometry" refers to a technique well known to those skilled in the art making it possible to scroll particles, molecules or cells at high speed in the beam of a laser, by counting them and by characterizing them. It is the light re-emitted by the particles, molecules or cells (by diffusion or fluorescence) which makes it possible to characterize them according to the desired criterion or criteria. Generally, the particles, molecules or cells are labelled with a fluorochrome which absorbs the energy of the laser and which reemits the energy absorbed in the form of photons of a higher wavelength. In the context of the invention, the reemitted light is obtained by a specific labeling of glycated hemoglobin present in red blood cells with an anti-glycated antibody, such as anti-HbAcl antibody, conjugated to a fluorochrome.

As used herein, the term "fluorochrome" (or "fluorophore") denotes a chemical substance capable of emitting fluorescence light after excitation by a laser. In the context of the invention, the fluorochrome is coupled to an anti -glycated antibody, such as anti-HbAcl antibody. Those skilled in the art have a wide choice of fluorochromes suitable for flow cytometry. All fluorochromes which can be coupled to an antibody can be used in the context of the present invention. In a particular embodiment, fluorochrome is fluorescein isothiocyanate (FITC), pacific bleu (PB), alexa fluor (AF), phycoerythrin (PE) or one of their derivatives, preferably PE. The PE, by its conformational properties (steric hindrance) has the advantage of having a PE: antibody ratio close to 1. The coupling chemistries of the antibodies are well known to those skilled in the art and the present invention is not limited to a particular coupling chemistry.

As used herein the term "antibody" or "immunoglobulin" have the same meaning, and will be used equally in the present invention. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen. As such, the term antibody encompasses not only whole antibody molecules, but also antibody antibody fragments comprising an antigen-binding domain (such as Fab, Fab’ and F (ab) 2, scFv, the fragments comprising either a VL domain or a VH domain), monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, primary antibodies, monospecific antibodies, multi-specific antibodies, single-chain antibodies (eg of camelid type). The antibodies according to the invention may be antibodies of any type, for example, IgG, IgE, IgM, IgD, IgA and IgY, of any class, for example, IgGl, IgG2, IgG3, IgG4, IgAl and IgA2 or any subclass. In the context of the present invention, the antibody is an antiglycated hemoglobin antibody, preferably an anti-HbAIc antibody, in particular an anti- HbAIc monoclonal antibody. As used herein, the term "sample" denotes a sample containing red blood cells obtained from a patient and from which it is possible to implement the invention. Advantageously, the sample is a blood sample, preferably a human blood sample. The sample can be taken from a patient at any time, for example by a blood intake. In the context of the invention, the sample has been taken before the methods of the invention.

As used herein, the term “hemolysis” also known as “erythrolysis” or “erythrocytolysis” refers to the lysis of red blood cells, leading to the release to hemoglobin into blood plasma.

As used herein, red blood cell-related disorder refers to a group of conditions that affect red blood cells and in particular that reduces their lifespan and include hemoglobin- related disorder; anemia; red blood cell enzyme deficiencies such as glucose-6-phosphate dehydrogenase deficiency and pyruvate kinase deficiency; red blood cell membrane disorders such as hereditary spherocytosis, hereditary elliptocytosis, hereditary pyropoikilocytosis, and hereditary stomatocytosis; myelodysplasia such as refractory anemia, refractory cytopenia with multilineage dysphasia and mixed myelodysplastic/myeloproliferative neoplasms and pathological conditions leading to destruction of red blood cell such as extracorporeal circulations, including ECMO and cardiac valvular disease.

As used herein, the term “hemoglobin-related disorder" also known as “hemoglobinopathies” has its general meaning in the art and refers to a group of inherited autosomal recessive pathologies affecting the Hb. Various abnormalities can result from changes (mutations or deletions) in the genes that regulate the production of Hb. Other mutations, even if they do not prevent the production of adequate levels of Hb chains, cause an alteration in their structure. They are known as hemoglobin variants and they alter the function and/or the stability of the entire Hb molecule and thus theirs lifespan. Hemoglobin-related disorder includes but are not limited to hemoglobinopathy such as thalassemia (such as a- Thalassemia or P-Thalassemia), Hemoglobin C disease, Hemoglobin S disease, Hemoglobin SC disease, Hemoglobin E disorders (or Hb E related syndroms), unstable abnormal Hb (such as Hb Taybe).

As used herein, the term “sickle cell disease” or “SCD” has its general meaning in the art and refers to an inherited blood disorder caused by a genetic mutation in the P-chains of hemoglobin, due to mutations in the HBB gene on chromosome 11 leading to the presence of abnormal HbS. The clinical expression of SCD is highly heterogeneous and there is broad inter- and intra-individual (during the patient’s life) variability, with phenotypes ranging from mild to severe disease with significant organ damage and early death. In SCD, these sickle cells also become rigid and sticky, which can slow or block blood flow results in a risk of various lifethreatening complications. The term includes sickle cell anemia (SCA) (hemoglobin SS or homozygous SS patient), hemoglobin SC disease and hemoglobin S/beta-thalassemia.

As used herein, the term “anemia” has its general meaning in the art and refers to a blood disorder occurring when there aren’t enough healthy red blood cells to carry oxygen. Anemia can be caused by blood loss, decreased red blood cell production, and increased red blood cell breakdown.

Anemias caused by an increased destruction of RBC are also known as “peripherical anemias” or “hemolytic anemias” and includes hereditary spherocytosis, hereditary elliptocytosis, abetalipoproteinemia, pyruvate kinase and hexokinase deficiencies, Glucose-6- phosphate dehydrogenase deficiency and glutathione synthetase deficiency, hemoglobinopathies such as sickle cell anemia, paroxysmal nocturnal hemoglobinuria, warm autoimmune hemolytic anemia, Cold agglutinin hemolytic anemia, Rh disease, transfusion reaction to blood transfusions, microangiopathic hemolytic anemias, including thrombotic thrombocytopenic purpura and disseminated intravascular coagulation, and hemodialysis.

Anemia caused by a decreased red blood cell production are also known as central anemia and includes pure red cell aplasia, aplastic anemia, anemia of kidney failure, anemia of endocrine disease, pernicious anemia, anemia of folate deficiency, anemia of prematurity, iron deficiency anemia, thalassemias, congenital dyserythropoietic anemias, myelophthisic anemia, myelodysplastic syndrome, anemia of chronic inflammation and leukoerythroblastic anemia.

As used herein, the term “subject” or “patient” refers to any mammal, such as a rodent, a feline, a canine, and a primate. Particularly, in the present invention, the term “subject” refers to a human and more particular, to a human afflicted with red blood cell-related disorder. In some embodiment, the subject is a human afflicted with anemia. In some embodiment, the subject is a human afflicted with sickle cell disease or beta-thalassemia and more particular with sickle cell anemia.

As used herein, the term "treatment" or "treat" refer to curative or disease modifying treatment, to cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of the red blood disorder-related disorder, such as anemia, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.

A “therapeutically effective amount” is intended for a minimal amount of active agent which is necessary to impart therapeutic benefit to a subject. For example, a "therapeutically effective amount" to a subject is such an amount which induces, ameliorates or otherwise causes an improvement in the pathological symptoms, disease progression or physiological conditions associated with or resistance to succumbing to a disorder. It will be understood that the total daily usage of the compounds used to treat red blood cell-related disorder will be decided by the attending physician within the scope of sound medical judgment.

1) Method for determining the relative age of subpopulations of red blood cells

In a first aspect, the invention related to an in vitro method for determining the relative age of at least two subpopulation of red blood cells of a set of red blood cells, comprising the steps of i) determining the level of glycated hemoglobin of each subpopulation of red blood cells of a set of red blood cells by flow cytometry, ii) comparing between them each of the glycated hemoglobin level determined in step i), and iii) concluding that the sous-population of red blood cells exhibiting a higher glycated hemoglobin level are older and that the sous- population exhibiting lower glycated hemoglobin level of red blood cells are younger.

In other words, the invention are suitable for determining the relative age of at least two subpopulation of red blood cells of a set of red blood cells, wherein, the level of glycated hemoglobin of a first subpopulation of red blood cells are compared with the level of glycated hemoglobin of an second subpopulation and it is concluded that said first subpopulation of red blood cells are older than said second subpopulation when the level of glycated hemoglobin of the first subpopulation of red blood cells is higher than the level of glycated hemoglobin of the second subpopulation.

In particular embodiment, regarding all the methods of the invention, the level of glycated hemoglobin of red blood cells are determined comprising the steps of : a) permeabilizing the membrane of isolated red blood cells, b) labelling the glycated hemoglobin of red blood cells obtained at step a) with an antiglycated hemoglobin antibody conjugated to a fluorochrome; c) measuring, by flow cytometry, the fluorescence intensity (FI) of each red blood cell, preferably at low speed acquisition, d) determining the glycated hemoglobin level (the geometric mean or median fluorescence intensity) of red blood cells.

In this particular embodiment, the glycated hemoglobin level is determined for each of the red blood cells of a set of at least 10,000 red blood cells, preferably at least 100,000 red blood cells, preferably at least 200,000 red blood cells, preferably between 50,000 and 200,000 red blood cells. In this particular embodiment, the red blood cells are previously isolated from a sample before the step a).

Many techniques are available to those skilled in the art to isolate red blood cells from a sample containing red blood cells. This step consists in purifying (or increasing the purity level) of red blood cells from a sample comprising red blood cells. This step makes it possible in particular to eliminate certain elements (eg platelets and white blood cells) likely to be present in the sample. These techniques are generally simple to implement and generally have no particular difficulty for those skilled in the art that will be able to adapt them for their implementation in the present invention. Mention may be made, for example, of centrifugation, chromatography techniques, density gradient fractionation. For example, (i) a blood sample is centrifuged, (ii) the pellet (containing red blood cells) is collected and (iii) a suitable isotonic buffer (eg, a phosphate buffer) is added to the pellet to obtain a suspension of red blood cells, the steps (i) to (iii) being able to be repeated one or more times in order to obtain the isolated red blood cells. Step b) consists in permeabilizing the membrane of the isolated red blood cells. This step consists in making the membrane of the red blood cells sufficiently permeable so that the anti -hemoglobin (HbF, HbS, HbAlc, etc...) antibody can penetrate into the red blood cells, while preserving the integrity of the red blood cells (without lysis of the red blood cells). Numerous techniques are available to those skilled in the art to permeabilize the red blood cell membrane. These techniques are generally simple to implement and generally have no particular difficulty for those skilled in the art that will be able to adapt them for their implementation in the present invention. Mention may be made, for example, of the use of a chemical agent such as a detergent and/or a surfactant (eg saponin, SDS and/or Triton).

In a particular embodiment, the membrane of the isolated red blood cells is fixed before the permeabilization step. The fixing of the membrane of the red blood cells makes it possible to avoid (or limit) the lysis of the red blood cells during the permeabilization step. Several chemical compounds are available to those skilled in the art to fix the membrane of the red blood cells. In a particular embodiment, the membrane of the isolated red blood cells is fixed with sodium azide and/or formaldehyde. Generally, the sodium azide and/or formaldehyde is simply added to a pellet of red blood cells or in a suspension of red blood cells.

In particular embodiment, the anti-glycated antibody conjugated to a flurorochrome is anti -HbAlc antibody, and more particularly an anti -HbAlc monoclonal antibody. In particular embodiment, the step of labelling the glycated-hemoglobin (GHb) is carried out under conditions allowing an intracellular binding between the anti-glycated hemoglobin antibody (anti-GHb antibody such as anti -Hb Ale antibody) and the glycated hemoglobin (such as HbAlc). This step makes it possible to obtain labeled red blood cells. Preferably, a sufficient quantity of anti-GHb antibodies is used to be able to mark the set of GHb, for example an excess of anti-GHb antibodies is used. That is, the bond between the anti- GHb antibody and the GHb takes place inside the red blood cell. Generally, the labeling consists in incubating the red blood cells whose membrane has been permeabilized with the anti-GHb for a time sufficient to allow an intracellular binding between the anti-GHb antibody and the GHb. The duration is generally a few minutes, for example between 5 min and 20 min. In a particular embodiment, the red blood cells are washed after the marking step. The washing makes it possible to eliminate the anti-GHb antibodies which would not be linked to GHb. The washing can be carried out with a suitable buffer, for example a PBS buffer. Washing also makes it possible to remove the chemical agent which has been used to permeabilize the red blood cell membrane. The red blood cells thus washed are then used in the flow cytometry measurement step.

Advantageously, the fluorescence intensity (FI) of each red blood cell of the set of red blood cells is measured independently of the fluorescence intensity (FI) of the other red blood cells of the set of red blood cells. In this step, a set of labeled red blood cells, that is to say all or part of the labeled red blood cells, is therefore analyzed by flow cytometry. Thus, the fluorescence intensity (FI) of each red blood cell of the set of red blood cells is measured independently of the fluorescence intensity (FI) of the other red blood cells of the set of red blood cells. The fluorescence intensity of each of the red blood cells taken independently is therefore measured at this step. In a particular embodiment, the fluorescence intensity (FI) is measured for each red blood cell of a set of at least 10,000 red blood cells, preferably at least 100,000 red blood cells, preferably at least 200,000 red blood cells, preferably between 50,000 and 200,000 red blood cells.

In particular embodiment, the fluorescence intensity (FI) of each red blood cell is measured at low-speed acquisition.

The last step consists in determining the glycated hemoglobin level of each red blood cell of the set of red blood cells. The HbAlc level of each red blood cell is determined from the fluorescence intensity measured by flow cytometry. The fluorescence intensity (FI) positively correlates with the level of glycated hemoglobin in red blood cell. As used herein, the term “level of glycated hemoglobin of set of red blood cells” refers to the value of mean fluorescence intensity (MFI) measure by flow cytometry, i.e the geometric mean or median fluorescence intensity of the set of red blood cells. As used herein, the term “level of glycated hemoglobin of the subpopulation of the set of red blood cells” refers to the value of mean fluorescence intensity (MFI) measure by flow cytometry, i.e the mean or median fluorescence intensity of the specific subpopulation of red blood cells.

In particular embodiment, the level of glycated hemoglobin of each RBC is normalized with the total level of hemoglobin per RBC, by applying the formula: GHb/Total Hb.

Fetal hemoglobin cannot be glycated. Thus, in order to determine the most accurate level of glycated hemoglobin in each RBC, it can be relative to the total of hemoglobin minus HbF content.

In particular embodiment, the level of glycated hemoglobin of each RBC can be normalized with the level of total hemoglobin per RBC minus the level of HbF per RBC , by applying the formula : GHb/(Total Hb - HbF).

Methods for determining the level of total hemoglobin per RBC are well known in the art. Hemoglobin determinations will usually be performed by an automated cell counter from a tube of well-mixed EDTA-anticoagulated blood filled to a predetermined level.

Subpopulation of red blood cells according to their glycated hemoglobin levels (i.e High HbG or Low HbG subpopulation) can also be identified with the method of the invention, each subpopulation of red blood cells exhibiting a different mean fluorescence intensity (MFI).

In particular embodiment, it is concluded that the subpopulation of red blood cells exhibiting a higher glycated hemoglobin level (high HbG) are older than the subpopulation exhibiting lower glycated hemoglobin level of red blood cells (lower HbG).

Thus, the invention also refers to a method for identifying subpopulation of red blood cells based on their relative age of a set of red blood cells in a subject comprising the steps of: i) determining by flow cytometry the level of glycated hemoglobin of red blood cells of the set of red blood cells from a sample obtained from said subject, ii) identifying a first subpopulation of red blood cells containing low level glycated hemoglobin (low GHb RBCs) and a second subpopulation of red blood cells containing high level glycated hemoglobin (high GHb RBCs), wherein the subpopulation of red blood cells exhibiting a higher glycated hemoglobin level (high HbG RBCs) are older than the subpopulation exhibiting lower glycated hemoglobin level of red blood cells (low HbG RBCs).

In particular embodiment, the subject has been blood transfused. In particular embodiment, when the subject has been blood transfused, the subpopulation of RBCs with high glycated hemoglobin (high GHb RBCs) corresponds to the healthy red blood cells transfused.

In some embodiments, the method determines the relative age of 2, 3, 4 or more subpopulation of red blood cells of a set of red blood cells.

In some embodiments, the subpopulations of red blood cells are based on their HbF levels and/or HbA and/or HbS levels.

In some embodiments, the subpopulation of red blood cells are based on their HbF levels (i.e high HbF RBCs and low HbF RBCs).

In some embodiments, the subpopulation of red blood cells are based on their HbA levels (i.e high HbA RBCs and low HbA RBCs).

In some embodiments, the subpopulation of red blood cells are based on their HbS levels (i.e high HbS RBCs and low HbS RBCs).

In some embodiments, the subpopulation of red blood cells are based on their genetically engineered hemoglobin levels. Such Hb are developed based on P-globin gene with specific amino acid substitution (i.e HbA ^T87Q [a2;P ^T87Q 2 -globin] in which the threonine at position 87 is replaced by a glutamine (T87Q) and HbAS3 [a2;P ^AS3 2-globin] in which the threonine at position 87 is replaced by a glutamine (87Q) the glutamic acid at position 22 is replaced by an alanine (A22) and the glycine at position 16 is replaced by an aspartic acid (016)).

In some embodiments, the subpopulation of red blood cells are based on their blood group surface antigen (i.e A, B, Rh, C, E, c, e, K, Jka, Jkb, Fya, Fyb, N, S, s).

In some embodiments, the subpopulation of red blood cells are based on their membrane protein expression levels (i.e Band 3, Ankyrin, Spectrin).

In some embodiments, the subpopulation of red blood cells are based on their intracellular protein expression levels (i.e glucose-6-phosphate dehydrogenase, pyruvate kinase, pyrimidine 5' nucleotidase).

In some embodiments, the subpopulation of red blood cells are based on their absolute HbF, HbA or HbS content quantified by flow cytometry (i.e red blood cells with more than 2, 4, 6, 8 and 10 pg).

Method for determining the level and absolute content of HbF, HbA, HbS, HbA2 in each red blood cells of a set of red blood cells are well known in the art and are described in US 2022/011324 Al. Techniques to identify statistically subpopulation of cells by single cell flow cytometry data analysis are well known in the art and are described in Pyne et al. PNAS, 2009 or Lampariello et al. Cytometry, 1998.

In particular embodiment, the level of glycated hemoglobin of each subpopulations of red blood cells based on their HbF and/or HbA2 and/or HbS levels are determined comprising the steps of : a) permeabilizing the membrane of isolated red blood cells, b) labelling the glycated hemoglobin of the red blood cell obtained at step a) with an anti-glycated hemoglobin antibody conjugated to a first fluorochrome capable of emitting a first fluorescence and; c) labelling the HbF of the red blood cells obtained in step a) with anti-HbF antibody conjugated to a second fluorochrome capable of emitting a second fluorescence and/or with anti-HbA antibody conjugated to a third fluorochrome capable of emitting a third fluorescence, and/or with anti-HbS antibody conjugated to a fourth fluorochrome capable of emitting a fourth fluorescence d) measuring, by flow cytometry, the fluorescence intensity (FI) of each fluorescence emitted by the first, and second and/or third fluorochrome of each red blood cell of the set of red blood cells; e) determining the level of HbF and/or of HbA and/or HbS (i.e fluorescence intensity emitted by the second or third or fourth fluorochrome) of each red blood of the set of red blood cells, f) identifying subpopulation of red blood cell based on the level of HbA and/or of HbF and/or of HbS, and g) determining the level of glycated hemoglobin of each subpopulation.

In particular embodiments, the red blood cells are previously isolated from a sample and the membrane of the red blood cells isolated are permeabilized before the step a).

2) Methods for diagnosis hemolysis and hemolytic anemia.

Chronic or acute hemolysis is defined as an imbalance between the rate of red blood cell (RBC) production and destruction. Severity of hemolysis is mainly appreciated through the total hemoglobin (Hb) concentration in blood measurement. Consequently, depending on the degree of importance of the imbalance, hemolysis results in low, moderate or severe anemia in patients. Whatever the cause, hemolysis results in an overall reduced lifespan of RBCs, as they are prematurely removed from the circulation.

Thus, the invention refers to an in vitro method for diagnosis hemolysis in a subject, comprising the step of i) determining the level of glycated hemoglobin of a set of red blood cells in a sample obtained from said subject by flow cytometry, ii) comparing the level of glycated hemoglobin determined at step i) with a reference value, and iii) concluding that the subject has a hemolysis when the level of glycated hemoglobin determined at step i) are lower than the reference value.

The method of the invention is also suitable to diagnosis hemolytic anemia.

Anemia is a serious global public health problem that particularly affects young children and pregnant women. WHO estimates that 42% of children less than 5 years of age and 40% of pregnant women worldwide are anemic. It is important to determine rapidly the causes of anemia, i.e an hemolytic anemia or anemia caused by a decreased red blood cell production in order to treat efficiently the subject. Hemolytic anemia will result in an overall reduced lifespan of RBCs while maintaining a normal red blood cell production.

Thus the invention also refers to an in vitro method for diagnosis hemolytic anemia in a subject, comprising the step of i) determining the level of glycated hemoglobin of red blood cells of a set of red blood cells in a sample from said subject by flow cytometry, ii) comparing the level of glycated hemoglobin determined at step i) with a reference value, and iii) concluding that the subject has a hemolytic anemia when the level of glycated hemoglobin determined at step i) are lower than the reference value.

In other words, the invention relates to an in vitro method for assessing a subject’s risk of having hemolytic anemia, comprising the steps of i) determining the level of glycated hemoglobin of red blood cells of a set of red blood cells in a sample from said subject by flow cytometry, ii) comparing the level of glycated hemoglobin determined at step i) with a reference value, and iii) concluding that the subject has a high risk of having hemolytic anemia when level of glycated hemoglobin determined at step i) are lower than the reference value.

In particular embodiment, regarding the method for diagnosis or for assessing a subject’s risk of having hemolytic anemia, the subject is afflicted with anemia caused by a decreased red blood cell production. In other words, the method is suitable to diagnose mixed anemia (i.e anemia caused by a decreased RBC production and an increased destruction of RBC). "Risk" in the context of the present invention, relates to the probability that the patients have or not an hemolytic anemia. Risk can be measured with reference to either actual observation post-measurement for the relevant cohort (i.e validation of the haemoglobin-related disorder with further measurement such as microcytic hypochromic anemia, nucleated red blood cells on peripheral blood smear,..), or with reference to index values developed from statistically valid historical cohorts._Odds ratios, the proportion of positive events (i.e having hemolytic anemia disorder) to negative events (i.e not having hemolytic anemia) for a given test result, are also commonly used (odds are according to the formula p/(l-p) where p is the probability of having hemolytic anemia and (1- p) is the probability of no having hemolytic anemia.

In some embodiments, lower level of glycated hemoglobin of red blood cells correlates to the severity of hemolytic anemia.

An additional object of the invention relates to an in vitro method for monitoring hemolytic anemia in a subject in need thereof comprising the steps of i) determining by flow cytometry the level of glycated hemoglobin of red blood cells of a set of red blood cells in a sample obtained from said subject at a first specific time, ii) determining by flow cytometry the level of glycated hemoglobin of red blood cells of a set of red blood cells in a sample obtained from said subject at a second specific time, iii) comparing the level of glycated hemoglobin determined at step i) with the level of glycated hemoglobin determined at step ii), and iv) concluding that the hemolytic anemia has evolved in worse manner when the level determined at step ii) is lower than the concentration determined at step i).

In some embodiments, the hemolytic anemia is sickle cell anemia.

In some embodiments, when method for diagnosis hemolytic anemia, it is concluded that the subject has an anemia caused by a decreased red blood cell production when the level of glycated hemoglobin determined at step i) are similar or not lower than the reference value.

In other words, the invention refers also to a method for classifying and/or stratifying subject afflicted with hemolytic anemia or anemia caused by a decreased red blood cell production comprising the steps of i) determining the level of glycated hemoglobin of a set of red blood cells in a sample from said subject, ii) comparing the level of glycated hemoglobin determined at step i) with a reference value, and iii) concluding that the subject has a hemolytic anemia when the level of glycated hemoglobin determined at step i) are lower than the reference value or concluding that the subject has an anemia caused by a decreased red blood cell production when the level of glycated hemoglobin determined at step i) are similar or not lower than the reference value.

In some embodiments, when the subject is diagnosed or classified as having hemolytic anemia, an appropriate treatment of RBC-related disorder is initiated, such as for example a treatment with hydroxyurea and/or erythropoietin.

In some embodiments, when the subject is classified as having hemolytic anemia, a therapy selected from the group consisting in hydroxyurea, voxelotor, HbF inducer treatment, erythroipoietin, , CD20 inhibitor such as ocrelizumab, rituximab, ofatumumab, ibritumomab, C5 inhibitor such as ecylizumab and crovalimab, C3 inhibitor such as pegcetacoplan, and Cl esterase inhibitor such as berinert and sutimlimab.

In some embodiments, when the subject is diagnosed as having hemolytic anemia, a therapy selected from the group consisting in hydroxyurea, voxelotor, HbF inducer treatment, erythroipoietin, , CD20 inhibitor such as ocrelizumab, rituximab, ofatumumab, ibritumomab, C5 inhibitor such as ecylizumab and crovalimab, C3 inhibitor such as pegcetacoplan, and Cl esterase inhibitor such as Berinert and Sutimlimab is administered to said subject and when the subject is diagnosed as having an anemia caused by a decreased red blood cell production, a therapy selected from the group consisting in bone marrow transplantation, corticosteroid, blood transfusion, immunoglobulins, and immunosuppressive therapy is administered to said subject.

In some embodiments, the reference value is a threshold value or a cut-off value. Typically, a "threshold value" or "cut-off value" can be determined experimentally, empirically, or theoretically. A threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognised by a person of ordinary skilled in the art. For example, retrospective measurement of the AHSP concentration in properly banked historical subject samples may be used in establishing the reference value. The threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. The full name of ROC curve is receiver operator characteristic curve, which is also known as receiver operation characteristic curve. It is mainly used for clinical biochemical diagnostic or prognostic tests. ROC curve is a comprehensive indicator that reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1 -specificity). It reveals the relationship between sensitivity and specificity with the image composition method. A series of different cut-off values (thresholds or critical values, boundary values between normal and abnormal results of diagnostic test) are set as continuous variables to calculate a series of sensitivity and specificity values. Existing software or systems in the art may be used for the drawing of the ROC curve, such as: MedCalc 9.2.0.1 medical statistical software, SPSS 9.0, ROCPOWER.SAS, DESIGNROC.FOR, MULTIREADER POWER. SAS, CREATE-ROC.SAS, GB STAT VIO.O (Dynamic Microsystems, Inc. Silver Spring, Md., USA), Stata/Se version 12.0 software (StataCorp LP, College Station, TX, USA), etc.

In some embodiments, the reference value is the level of glycated hemoglobin of a set of red blood cells in a healthy subject (i.e that has not been diagnosed for a red blood cell- related disorder or anemia).

In some embodiments, the reference value is determined from the level of glycated hemoglobin of a set of red blood cells from one or more healthy subject (i.e that has not been diagnosed for a red blood cell-related disorder or anemia).

In some embodiments, the reference value is determined from the level of glycated hemoglobin in red blood cells of a set of red blood cells from one or more subject afflicted with anemia caused by a decreased red blood cell production.

In some embodiments, the reference value is the level of glycated hemoglobin of a subpopulation of red blood containing high levels of HbF (High HbF-RBCs population cells) in a healthy subject.

Furthermore, retrospective measurement of the level of the markers of the invention in properly banked historical subject samples may be used in establishing these reference values.

3) Methods for monitoring the efficacy of treatment of red blood cell-diseases.

Therapy of red blood cell-related disorders such as sickle cell disease or betathalassemia tends to increase lifespan of red blood cells (and thus increase of glycated hemoglobin) over time. Determined the relative age between a set of blood cells obtained from patient before and/or different time of treatment will allow to evolution of the therapeutic efficacy of said treatment. Thus, accordingly, the invention also relates to an in vitro method for monitoring the therapeutic efficacy of a treatment of red blood cell (RBC)-related disorder in a subject in need thereof comprising the steps of: i) determining by cytometry the level of glycated hemoglobin of a set of red blood cells of a sample from said subject at a first specific time, ii) determining by cytometry the level of glycated hemoglobin of a set of red blood cells of a sample from said subject at a second specific time of said treatment, iii) comparing the level of glycated hemoglobin determined at step i) with the level of glycated hemoglobin determined at step ii), and iv) concluding that the treatment of RBC-related disorder is efficient when the level of glycated hemoglobin determined at step ii) is higher than the level of glycated hemoglobin determined at step i).

In some embodiments, when it is concluded that the treatment is efficient, the treatment is continued.

In some embodiments, regarding the methods of the invention for monitoring the treatment of RBC-related disorder, it is concluded that said treatment is not efficient when the level of glycated hemoglobin determined at step ii) is similar or not higher than the level of glycated hemoglobin determined at step i).

In some embodiments, when it is concluded that the treatment exhibits not effect anymore or is not efficient, the treatment is adapted/modified.

According to the invention, adapting the treatment of RBC-related disorders refers to adjust/increase the dosage of the compounds used to treat RBC-related disorders and/or use an another compounds or therapy to treat RBC-related disorders.

In other words, the invention refers to a treating RBC-related disorder or monitoring the treatment of RBC-related disorder in subject in need thereof comprising the steps of i) determining by cytometry the level of glycated hemoglobin of a set of red blood cells of a sample obtained from a patient at a first specific time, ii) determining by cytometry the level of glycated hemoglobin of a set of red blood cells of a sample obtained from a patient at a second specific time of said treatment, iii) comparing the level of glycated hemoglobin determined at step i) with the level of glycated hemoglobin determined at step ii), and iv) continuing the treatment of RBC-related disorder when the level of glycated hemoglobin determined at step ii) is higher than the level of glycated hemoglobin determined at step i) or adapting the treatment of RBC-related disorder when the level of glycated hemoglobin determined at step ii) is similar or not higher than the level of glycated hemoglobin determined at step i).

In some embodiments, regarding the methods for treating or monitoring of the invention, the RBC-related disorder is a RBC-related disorder reducing the RBC lifespan.

In some embodiments, regarding the methods for treating or monitoring of the invention, the RBC-related disorder is a hemoglobin-related disorder.

In some embodiments, regarding the methods for treating or monitoring of the invention, the RBC-related disorder is sickle-cell disease or beta-thalassemia.

In some embodiments, regarding the methods for treating or monitoring of the invention, the RBC-related disorder is sickle cell disease such as sickle cell anemia.

In some embodiments, regarding the methods for treating or monitoring of the invention, the level of glycated hemoglobin determined at step i) is determined before or at the beginning of the treatment of RBC-related disorder.

In some embodiments, regarding the methods for treating or monitoring of the invention, the level of glycated hemoglobin determined at step i) is determined at a first specific time of the treatment of RBC-related disorder.

In the context of the invention, the term “treatment of RBC-related disorder” refers to therapy, such as gene therapy, bone marrow transplantation (to correct deficiencies in the synthesis or structure of hemoglobin chain for example or to increase hemoglobin level), blood transfusion or compounds used to treat RBC-related disorder such as sickle cell disease and/or beta-thalassemia. The compounds used to treat RBC-related disorder such as sickle cell disease or beta-thalassemia includes but are not limited to decitabine; decitabine/tetrahydouridine; panobinostat; pomalidomide; luspatercept; vepoloxamer; L-glutamine; voxelotor; erythropoiesis stimulating agents (ESA) such as erythropoietin (EPO), epoetin alfa, darbepoetin alfa and crizanlizumab; cobalamin; y chain synthesis stimulating agent; iron and iron chelation therapy such as deferoxamine, deferiprone and deferasirox; pyruvate kinase activators such as etavopivat and mitapivat; CD20 inhibitor such as ocrelizumab, rituximab, ofatumumab, ibritumomab, C5 inhibitor such as ecylizumab and crovalimab, C3 inhibitor such as pegcetacoplan, Cl esterase inhibitor such as Berinert and Sutimlimaband HbF inducer treatment. HbF inducer treatment has its general meaning in the art and includes gene therapy and HbF inducer compounds such as hydroxycarbamide (or hydroxyurea) sodium 2,2- dimethylbutyrate (NCT01322269) , lysine-demethylase 1 (LSD-1) inhibitor (NCT03132324), Stimulator of Soluble Guanylate Cyclase (sGC) (NCT03285178) , phosphodiesterase 9 inhibitor (NCT03401112), DNA-methyl transferase inhibitor (5-azacytidine, decitabine, GSK3482364), Histones deacetylase inhibitor (panobinostat, vorinostat, UNC0638), Immunomodulator (thalidomide, pomalidomide), Dimethyl fumarate (DMF) and metformin. Gene therapy also induces HbF expression in red blood cell using gene addition strategy to add y-globin (HbF) gene or through repression of the y-globin gene inhibition by targeting BCL11A either with RNA-silencing or with CRISPR-Cas9 induced gene disruption. Creation of deletions responsible for hereditary persistence of fetal hemoglobin (HPFH) were also described as HbF inducers and represent potent strategies in gene therapy.

In some embodiments, the treatment of RBC-related disorder is a compound used to treat RBC-related disorder.

In some embodiments, the treatment of RBC-related disorder is HbF inducer treatment.

To monitor more specifically HbF inducer treatment, it is relevant to determine the level of glycated hemoglobin in subpopulation of red blood cells exhibiting high levels of HbF.

Thus, in particular embodiment, regarding the methods for monitoring HbF inducer treatment, the level of glycated hemoglobin are determined in a high-HbF red blood cells subpopulation at step i) and step ii).

Thus, in particular embodiment, the invention also relates to an in vitro method for monitoring the therapeutic efficacy of HbF inducer treatment in a subject in need thereof comprising: i) determining at a first specific time the level of glycated hemoglobin of high-HbF red blood cells of a sample from a patient by cytometry, ii) determining at a second specific time of said treatment the level of glycated hemoglobin of high-HbF red blood cells of a sample from a patient, iii) comparing the level of glycated hemoglobin determined at step i) with the level of glycated hemoglobin determined at step ii), and iv) concluding that HbF inducer treatment is efficient when the level of glycated hemoglobin determined at step ii) is higher than the level of glycated hemoglobin determined at step i). In particular embodiment, the HbF inducer treatment is continued when the level of glycated hemoglobin determined at step ii) is higher than the level of glycated hemoglobin determined at step i). In particular embodiment, the HbF inducer treatment is stopped and a new treatment of RBC-related disorder are used when the level of glycated hemoglobin determined at step ii) is similar or not higher than the level of glycated hemoglobin determined at step i). In particular embodiment, HbF inducer treatment is adapted (such as increase of dose) when the level of glycated hemoglobin determined at step ii) is similar or not higher than the level of glycated hemoglobin determined at step i).

In some embodiments, the HbF inducer treatment is hydroxycarbamide.

As used herein, the term “hydroxy carbamide”, or “HC”, also known as “hydroxyurea” has its general meaning in the art and refers to a compounds having the following formula CH4N2O2 currently used to treat sickle-cell disease and/or beta-thalassemia, chronic myelogenous leukemia, or cervical cancer. Its CAS number is 127-07-1. HC treatment is often the first choice in SCD adult patients with symptomatic disease, to avoid transfusion and the risks it entails, and in children, to prevent further complications of the disease. However, the response to HC is highly variable and differs from one patient to another. HC has been shown to increase HbF levels, thereby decreasing the rate of HbS polymerisation.

In some embodiments, the treatment of RBC disorder is blood transfusion.

Blood transfusion of healthy’ s subject is used to provide normal red blood cells to the subject afflicted with a RBC-related disorders, such as sickle cell disease or beta-thalassemia. These are two types of red blood cells transfusions : simple transfusion, where additional healthy red blood cells are transfused to the subject’s body and exchange transfusions where the patient’s sickle-shaped blood cells are exchanged with healthy ones. In both cases, the age and lifespan of the red blood cells of the patients must increase after the transfusion. Some subject need transfusions every few months whilst others need one every couple of weeks. The methods of the invention are suitable to monitor the blood transfusion and determine if the subject need a new blood transfusion.

In particular embodiment, when method monitoring blood transfusion in subject in need thereof, the level of glycated hemoglobin determined at step i) is determined before the blood transfusion.

Thus, in particular embodiments, the invention also relates to an in vitro method for monitoring the therapeutic efficacy of blood transfusion in a subject in need thereof comprising: i) determining before blood transfusion the level of glycated hemoglobin of a set of red blood cells of a sample from a patient by cytometry, ii) determining at a second specific time after blood transfusion the level of glycated hemoglobin of a set of red blood cells of a sample from a patient by cytometry, iii) comparing the level of glycated hemoglobin determined at step i) with the level of glycated hemoglobin determined at step ii), and iv) concluding that blood transfusion exhibits still an effect when the level of glycated hemoglobin determined at step ii) is higher than the level of glycated hemoglobin determined at step i).

In particular embodiment, when it is concluded that blood transfusion does not exhibit effect anymore, a new blood transfusion can be performed.

FIGURES:

Figure 1: A. Differences in HbAlc levels between AA and SS patients in whole blood. Levels were measured by HPLC or by immunologic DCA method for AA and SS, respectively. (Mann-Whitney test). B. Differences in HbAlc levels between AA and SS patients in whole circulating RBCs. Levels were measured by flow cytometry after intracellular staining of HbAlc using a monoclonal fluorescent antibody. (Mann-Whitney test).

Figure 2: In vitro glycation of HbA in purified RBCs from healthy donors and incubated in PBS pH = 7.4 supplemented with 10 g/L of D-glucose. RBCs were collected at day 0, 3 and 7 and analysed by FACS for HbAlc MFI measures after fixation, permeabilization and intracellular staining using a monoclonal fluorescent antibody. Data are mean ± standard deviation (n = 5). Pearson test.

Figure 3: A. Representative flow cytometry histogram of a transfused SS patient displaying two distinct population in term of HbS content within RBCs : the transfused RBCs, HbS-negative population (left) and the patient’s own RBCs, HbS-positive population (right). HbS distribution was obtained by flow cytometry after intracellular staining using a monoclonal fluorescent antibody. B. Differences in HbAlc levels between transfused donor RBCs (HbS- negative) and patient’s own RBCs (HbS-positive) assessed in transfused SS patients. Levels were measured by flow cytometry after intracellular staining of both HbS and HbAlc using a monoclonal fluorescent antibody.

Figure 4. Representative flow cytometry histogram of a transfused SS patient displaying two distinct population in term of GHb content within RBCs : the transfused RBCs, GHb-high population (right) and the patient’s own RBCs, GHb-low (left). GHb distribution was obtained by flow cytometry after intracellular staining using a monoclonal fluorescent antibody.

Figure 5: A. Representative flow cytometry histogram of a SS patient displaying a heterogeneous distribution of HbF in RBCs. HbF was assessed by flow cytometry after intracellular staining using a monoclonal fluorescent antibody. Two sub-populations were selected according to HbF fluorescence intensity : HbF-Low and HbF-High. B. Differences in HbSlc levels between HbF-low and HbF-High subpopulations in SS patients. Levels were measured by flow cytometry after intracellular staining of both HbF and HbAlc using monoclonal fluorescent antibodies. (Wilcoxon test). C. Differences in HbAlc levels between HbF-low and HbF-High subpopulations in AA patients. Levels were measured by flow cytometry after intracellular staining of both HbF and HbAlc using monoclonal fluorescent antibodies. (Wilcoxon test).

EXAMPLE:

Material & Methods

Participants. The study was conducted in compliance with the Declaration of Helsinki and approved by the local Institutional Review Board (CPP - Creteil). All participants gave a written informed consent to blood donation and their data rendered anonymous to protect patients’ privacy and confidentiality.

Exclusion criteria were individuals with diabetes or pre-diabetes.

Every blood samples were collected by venipuncture using heparin or EDTA as anticoagulant and were processed within 24h after blood withdrawal.

RBC parameters. RBC parameters including mean cell volume (MCV), mean corpuscular hemoglobin content (MCH) and mean corpuscular hemoglobin concentration (MCHC) were determined using ABX Micros ES 60 Hematology Analyzer (HORIBA Medical).

HbAlc and HbF dosage in RBC hemolysates. To measure HbAlc, two techniques were performed: cation-exchange high-pressure liquid chromatography (HPLC) and a rapid glycohaemoglobin (HbAlc) immunoassay (DCA).

HPLC. An automated HPLC system (VARIANT™ II, BIO-RAD, HbA2/HbAlc dual program) was used to quantify the level of HbF in all participant RBC hemolysates as well as the level of HbAlc containing in only healthy subject (Hb AA) RBC hemolysates. Experimental conditions were as recommanded by the manufacturer. DCA test HbAlc containing in RBC hemolysates provided from participants with the variants of hemoglobin (Hb SS, Hb AS, Hb SC) was determined by DCA-Vantage point-of- care benchtop analyzer (Siemens).

HbAlc and HbF level estimation in individual RBCs by flow cytometry.

Cell fixation and permeabilization. RBC fixation and permeabilization were performed with Fetal Cell Count™ Kit reagents (IQ Products bright fluorescence) following the manufacturer’s instructions.

Cell staining by immunofluorescence. Fixed and permeabilized RBCs were incubated for 15 min at room temperature in the dark with a mixture containing mouse monoclonal antihuman HbAlc antibody (Biorbyt, Explore Bioreagents, HbAlc antibody orb 195572 unconjugated, Uniprot ID: P69905) conjugated with Alexa Fluor 647 (Alexa Fluor™ 647 Protein Labeling Kit, Thermo Fisher Scientific), HbF antibody conjugated with Phyco-Erythrin (PE) (reagent F, Fetal Cell Count™ Kit, IQ products). For each RBC fraction, two negative controls were used. The first was prepared with an isotype mixture containing Alexa Fluor 647 mouse IgGl kappa (BD Phosflow, cat 557783) and PE mouse IgGl kappa (BD Pharmingen, cat 555749) and the second one contained only fixed and permeabilized RBCs. After staining, RBCs were washed three times in PBS at 300 g for 3 min at room temperature and analyzed by flow cytometer using a BD FACS Canto II System (BD Biosciences).

For transfused patients, fixed and permeabilized RBCs were incubated with a mixture containing mouse monoclonal anti -human HbAlc antibody conjugated with Alexa Fluor 647 (Alexa Fluor™ 647 Antibody Labeling Kit, Thermo Fisher Scientific), and anti-human HbS antibody conjugated with Pacific blue (PB) (Antibody Labeling Kit, Thermo Fisher Scientific).

Flow cytometry acquisition. Alexa Fluor 647 and PEfluorescence intensities were monitored with PMT voltages set at 400 and 400 V, respectively. A minimum of 100,000 events were recorded. Data were analyzed with the FlowJo cell analysis software v.10. (FlowJo, Miltenyi Biotec).

In vitro glycation of hemoglobin. Purified RBCs from healthy donors where resuspended at 50% hematocrit in 0.9% NaCl solution supplemented with D-dextrose at a final concentration of 10 g/L. RBCs were kept at +20°C under low agitation for the indicated time. Hb was measured by flow cytometry.

Results The glycated Hb was significantly lower in SS patients than in healthy donors (glycated HbSS =3.7%, glycated HbAA =5.3%, p=0.0025 by DCA and HPLC respectively, and by flow cytometry p=0.0061) (Figure 1A and IB).

RBCs, kept in isotonic solution supplemented with 10 g/L of D-dextrose, exhibited increased levels of glycated Hb fluorescence intensity as compared to fluorescence measured at day 0 in a time dependent manner over 7 days (Figure 2).

In transfused SS patients, results showed that glycated Hb was lower in autologous SS- RBCs than in AA-RBCs by a factor of four (P= 0.008), thus confirming the characterized reduced lifespan of SS RBCs (Figure 3A-B). Representative flow cytometry histogram of a transfused SS patient displays two distinct population in term of glycated hemoglobin (GHb) level within RBCs : the transfused RBCs, GHb-high population and the patient’s own RBCs, GHb-low (Figure 4).

Glycated Hb levels were also measured in RBC sub-populations based on their HbF level (high HbF or low HbF subfractions) in non-transfused SS patients (Figure 5A). Glycated Hb level was lower in low HbF RBCs, as compared to high HbF RBCs (Figure 5B). This difference was not detected in healthy donors, thereby indicating that HbF has an effect on relative age only in SCD patients (Figure 5C).

Single cell glycated Hb assessed by flow cytometry allows to simultaneously estimate the relative age of different RBC sub-populations. This new tool is noninvasive and requires only one single time point measurement.

REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

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