METHOD FOR DETERMINING THE HAEMOGLOBIN CONTENT OF AN ERYTHROID CELL
20220011324 · 2022-01-13
Inventors
- Marie CAMBOT (L'Hay-les-Roses, FR)
- Gaetana Vandemeulebrouck (Paris, FR)
- France Noizat Pirenne (Paris, FR)
- Pablo Bartolucci (L'Hay-les-Roses, FR)
- Marie Georgine Rakotoson (Antananrivo, MG)
- Frédéric Galacteros (Santeny, FR)
- Nicolas Hebert (Ollainville, FR)
Cpc classification
G01N33/72
PHYSICS
A61P7/00
HUMAN NECESSITIES
International classification
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.
Claims
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.sup.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.sub.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.sup.th hemoglobin Hbxn of the erythroid cells obtained in step b) with at least one anti-Hbxn antibody conjugated to a n.sup.th fluorochrome capable of emitting a n.sup.th fluorescence, d.sub.1) measuring, by flow cytometry, the fluorescence intensity of each fluorescence emitted by the first, the second, the n.sup.th fluorochrome of each erythroid cell of a set of erythroid cells; e.sub.1) determining the content of the at least one first hemoglobin Hbx1, the at least one second hemoglobin Hbx2, the at least one n.sup.th hemoglobin Hbxn in each erythroid cell of the set of erythroid cells by comparing each of the first, the second, the n.sup.th fluorescence intensities measured in step d.sub.1) with a first, a second and an n.sup.th standard curve associating the measured first, second, and n.sup.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.sup.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.sub.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.sub.1) measuring, by flow cytometry, the fluorescence intensity (MFI) of each erythroid cell of a set of erythroid cells; e.sub.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.sub.1)-e.sub.1) for each of the at least one second Hbx2 (steps c.sub.2)-e.sub.2)), at least one third Hbx3 (steps c.sub.3)-e.sub.3)) and n.sup.th Hbxn (steps c.sub.n)-e.sub.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 containing red blood cells from a patient; b) determining the at least one hemoglobin Hbx content selected from the group consisting of the HbF, HbA and HbS content of each red blood cell of a set of red blood cells of said sample according to the method of claim 12; c) when at least a predetermined percentage of the red blood cells of the set of red blood cells has an HbS content which exceeds a HbS reference threshold, and/or when at least a predetermined percentage of the red blood cells of the set of red blood cells has an HbF and/or HbA content is below HbF and/or HbA reference threshold initiating an appropriate treatment in the patient.
17. The method as claimed in claim 16, wherein at least 20% of the red blood cells of the set of red blood cells have an HbF content which is below the reference threshold.
18. The method as claimed in claim 17, wherein the reference threshold is 2 pg or more.
19. The method as claimed in claim 17, wherein the reference threshold is selected from the group consisting of 3 pg, 4 pg, 5 pg, 6 pg, 7 pg, 7.5 pg.
20. The method as claimed in claim 16, wherein the suitable treatment is hydroxyurea and/or erythropoietin.
21. The method as claimed in claim 3, wherein the blood sample is a human blood sample.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0262]
[0263]
[0264] The signal measured is the area under the curve (A for area) of the variations in fluorescence intensities of each cell. Along the ordinate: red blood cell count.
[0265]
[0266]
[0267] The selection of the red blood cell singlets was carried out on the height and width parameters of the FSC signal first, then on the height and width parameters of the SSC signal.
[0268]
[0269]
[0270]
[0271]
[0272]
[0273]
[0274]
[0275]
[0276]
[0277]
[0278]
[0279]
[0280]
EXAMPLES
Example 1: Development of a Method for Determining the HbF Content of Each Red Blood Cell: Construction of a Standard Curve
[0281] A group of patients having a perfectly homogeneous distribution of the HbF content of each red blood cell was selected from the SICLOPEDIE collection monitored at the Unite de Maladies Génétiques du Globule Rouge [Red Blood Cell Genetic Diseases Unit] at the Centre Hospitalier Universitaire Henri Mondor [Henri Mondor University Hospital Center] in Créteil. This group is composed of adult patients (age≥18 years old) having a hereditary persistence of HbF (HPFH) or intermediate or minor β-thalassemia, and an HbF content of each of their red blood cells which is homogeneous and constant over time. The HbF content of the total red blood cells of patients was determined, in the context of patient monitoring, by regular measurement of the % HbF (by HPLC) and of the mean HbF content per red blood cell (MCHbFCo). Patients having a major sickle cell syndrome (SS, SB-Thalassemia or SC) or a sickle cell trait (AS) and also patients treated with an HbF inducer (Hydroxyurea), patients having been transfused during the past 3 months before inclusion and pregnant patients were not included.
[0282] In accordance with the Declaration of Helsinki on the ethical principles applicable to medical research involving human beings (World Medical Association, text in force 2008, paragraph 24), all selected patients were informed of the risks and benefits of this study and provided written consent before inclusion. In accordance with the legislation in force (Articles L.1121-3 and R.5121-13 of the French Public Health Code), the information relating to the patients was protected anonymously in order to ensure confidentiality throughout the duration of the study.
[0283] This research protocol was approved by the International Review Board Île-de-France IV located at the Hospital Saint-Louis (IRB No. 00003835).
[0284] In summary, 20 ml of blood were taken from the selected patients and collected in 5 tubes containing EDTA. The samples were treated within 24 h after having been taken: determination of the mean HbF concentration (using HPLC), blood hemogram, and flow cytometry analysis then freezing of the remaining samples at −80° C. for the subsequent analyses.
[0285] The detailed protocol is explained below.
[0286] 1. Pre-Treatment of the Samples
[0287] 20 ml of blood were taken from the selected patients and collected in 5 tubes containing EDTA. The red blood cells were recovered after fractionation of the whole blood by centrifugation at 800 G for 10 minutes at ambient temperature. Approximately 8 ml of blood cell pellet were collected and then washed in 10 ml of phosphate buffer (DPBS 1× Gibco by Life Technologies Cat 14190-094, Life Technologies SAS, Saint Aubin, France) in a 50 ml polypropylene tube. After homogenization, the blood cell suspensions were centrifuged at 1200 G for 5 minutes at ambient temperature. The blood cell pellet was recovered after elimination of the supernatant, and the washing procedure was carried out 3 times.
[0288] A first measurement of the HbF content (% HbF by HPLC×MCHCo) was carried out on approximately 1 ml of fresh sample, while 7 ml of blood cell pellet were stored at −80° C. in aliquot fractions of 200 μl in cryotubes (Nalgene Cat 479-6841) using glycerol (B Braun formula No. 569) as cryoprotective agent. For that, approximately 57.7% (V/V) of glycerol were added to the blood cell pellets in 2 steps. Specifically, in order to prevent precipitate formation, ⅓ of the glycerol volume was added dropwise to the blood cell pellet, in a first step, while agitating the cryotube, followed by an incubation for 10 minutes at ambient temperature, and the glycerol volume was subsequently made up with the remaining ⅔. With this procedure, the red blood cells can be stored for several months, or even years, while at the same time limiting lysis thereof.
[0289] The thawing consists of an incubation of the red blood cells in a bath at 37° C. or of rapid agitation in the hand, followed by a series of washing using 2 solutions of NaCl in decreasing concentrations in order to remove the glycerol and to preserve the isotonicity. For that, 75 μl of deglycerolization solution (12% NaCl, B Braun formula No. 570 Melsungen, Germany) were added dropwise in a first step while homogenizing the blood cell suspensions, then the suspensions were incubated at ambient temperature for 10 minutes. Subsequently, 125 μl of NaCl at 0.9% containing glucose were added and the suspensions were incubated for 5 minutes at ambient temperature. Approximately 4×125 μl of NaCl at 0.9% containing glucose were added while observing an incubation of 5 minutes between each volume of NaCl at 0.9% containing glucose. The blood cell suspensions were transferred into 2 ml Eppendorf tubes, then fractionated by gentle centrifugation at 300 G for 10 minutes at ambient temperature while applying a deceleration equal to 5. After removal of the supernatant, 4×125 μl of NaCl at 0.9% containing glucose were added while observing an incubation of 5 minutes between each volume of NaCl at 0.9% containing glucose, then the suspensions were centrifuged as previously. In order to obtain a dry pellet of red blood cells, 500 μl of NaCl at 0.9% containing glucose were added in one step, followed by a centrifugation at 800 G for 10 minutes at ambient temperature. At the end of the final certification, the cell samples can be brought to the desired hematocrit by making up the volume with a buffer solution.
[0290] 2. Determination of the HbF Content of Each Red Blood Cell
[0291] The determination of the HbF content of each red blood cell of the samples from selected patients was carried out both in the fresh cell samples and in the thawed samples using the following 3 methods: [0292] high-performance liquid chromatography (HPLC) to determine the mean HbF percentage over all of the red blood cells [0293] blood hemogram (including the mean MCHCo); and [0294] flow cytometry.
[0295] 2.1. Determination of the Mean HbF Percentage by HPLC
[0296] The determination of the mean HbF percentage by HPLC in the blood cell suspensions was carried out on a Variant II system (Cat: 2702000 Hemoglobin Testing System, Bio-Rad Laboratories, Marnes-la-Coquette, France). It is an ion exclusion liquid chromatography which makes it possible to separate 3 types of hemoglobins: HbF, HbA and HbA2 by virtue of the V2_B-THAL_DU method. The system is composed of a stationary phase composed of resin to which negatively charged carboxyl groups are attached and of a mobile phase composed of two Bis/Tris phosphate buffer solutions having a low and a high ionic strength respectively, and also of a photometric detector emitting 2 different wavelengths: 690 nm and 415 nm for detecting the negative control and the samples respectively. This system makes it possible to quantify the 3 hemoglobins from total blood or from a blood cell suspension with a minimum volume of 500 μl. For a sample of less than 500 μl, a dilution to 1:200 (v/v) with a dilution reagent was carried out before proceeding with the analysis. The analytes were separated as a function of their ionic interaction with the carboxyl groups over a total period of 6 minutes. The HbF was eluted after approximately 0.5 min. The data acquisition was carried out on a CDM 5.2 program in order to determine the mean percentages of each type of Hb. The mean HbF percentage is obtained according to the following relationship:
% HbF=(AUC HbF/AUC total Hb)×100
where % HbF represents the mean HbF percentage over all of the erythrocyte cells; AUC HbF represents the area under the curve of the HbF on the chromatogram; AUC total represents the sum of the areas under the curves of the 3 different types of Hb, corresponding in these patients to HbA, HbA2 and HbF.
[0297] 2.2. Determination of the Mean Hemoglobin Content Per Red Blood Cell (MCHCo)
[0298] The mean hemoglobin content per red blood cell (or mean corpuscular hemoglobin content or MCHCo) constitutes a blood cell constant obtained by complete blood count or blood hemogram. The MCHCo is determined by calculation, by dividing the hemoglobin concentration expressed per liter of blood by the number of red blood cells per liter of blood.
[0299] This analysis was carried out on a Horiba ABX Micros ES 60 automated counter (HORIBA Medical, Montpellier, France) on a minimum volume of sample of 500 μl having a hematocrit at approximately 50%.
[0300] 2.3. Determination of the Mean HbF Content Per Red Blood Cell (MCHbFCo)
[0301] The determination of 2 mean parameters (% HbF and MCHCo) obtained by HPLC and by blood hemogram made it possible to calculate the mean HbF content per red blood cell, or MCHbFCo, according to the following relationship: MCHbFCo=(% HbF×MCHCo)/100 where MCHbFCo (pg) represents the mean HbF content per red blood cell;
[0302] % HbF represents the mean HbF percentage determined by HPLC (see 2.1);
[0303] MCHCo represents the mean Hb content per red blood cell provided by the automated counter (see 2.2).
[0304] 3. Determination of the Fluorescence of the Red Blood Cells Labeled with an Anti-HbF Antibody by Flow Cytometry
[0305] 3.1. Instruments
[0306] The analysis of the HbF content of each red blood cell was carried out on an 8-color BD FACS Canto II cytometer (Cat 338960, BD Biosciences, Le Pont de Claix, France) combined with the DIVA® data acquisition software.
[0307] Flow cytometry or FACS (fluorescence-activated cell sorting) is a technique which makes it possible to individually analyze the properties (size, granulosity and fluorescence) of particles (beads or cells) in suspension in a flow system.
[0308] It is an immunological method based on the immunophenotyping technique which consists of an antigenic detection by virtue of recognition by a specific antibody which is usually conjugated to a fluorescent molecule. The signal measured is principally the fluorescence intensity, which is proportional to the amount of antigens detected by the specific antibody. The cytometry data acquisition requires pre-processing of the samples, which consists in fixing and permeabilizing the cell membrane, followed by intracellular labeling with an HbF-specific antibody conjugated to a fluorochrome.
[0309] 3.2. Fixing and Permeablilizing of the Red Blood Cell Membrane
[0310] The fixing and permeabilizing of the RBC membrane was carried out using the reagents of a kit for detecting F cells called “Fetal Cell Count™ kit” (Cat IQP-349, IQ Products, Groningen, the Netherlands).
[0311] In order to fix the blood cell membrane, 5 μl of washed blood blood cell pellet (fresh or frozen) were added and homogenized in 100 μl of fixing reagent or reagent A containing a preservative or sodium azide (Fetal Cell Count™ kit, Cat IQP-349). 100 μl of buffered formaldehyde solution or fixing solution B (Fetal Cell Count™ kit, Cat IQP-349) were subsequently added to the blood cell suspension. The suspension was homogenized by vortexing and then incubated at ambient temperature for exactly 30 minutes. The blood cell suspension was carefully homogenized every 10 minutes. The RBCs were subsequently washed in 2 ml of 1×PBS buffer containing heparin (reagent D Fetal Cell Count.sup.TM kit, Cat IQP-349), followed by centrifugation at 300 G for 3 minutes at ambient temperature. The washed blood cell pellet was resuspended by adding 100 μl of 1×PBS buffer (reagent D Fetal Cell Count™ kit, Cat IQP-349).
[0312] In order to permeabilize the blood cell membrane, 100 μl of sodium dodecyl sulfate solution, permeabilization reagent (reagent C Fetal Cell Count™ kit, Cat IQP-349) were added to the suspension of fixed RBCs. The samples were incubated for exactly 3 minutes at ambient temperature. The RBCs were washed as previously. The wash was carried out twice. After removal of the supernatant, the blood cell pellets were resuspended in 1 ml of 1×PBS buffer (reagent D Fetal Cell Count™ kit, Cat IQP-349).
[0313] 3.3. Intracellular Labeling of the Fixed and Permeabilized Red Blood Cells
[0314] An IgG1 Kappa clone mouse monoclonal antibody directed specifically against the gamma chain of human HbF and conjugated with phycoerythrin (PE) (reagent F Fetal Cell Count™ kit, Cat IQP-349, IQ Products) was used to determine the HbF content of each previously fixed and permeabilized red blood cell. For that, 50 μl of antibody solution were deposited in 50 μl of suspension obtained in 3.2. Phycoerythrin, by virtue of its conformational (steric hindrance) properties has the advantage of having a PE:antibody ratio close to 1.
[0315] A sample not labeled with the antibody was used as a negative control, while an isotype control was prepared by adding 20 μl of PE-coupled mouse IgG1 Kappa (Cat: 555749, BD Pharmingen™, Le Pont De Claix, France) in 50 μl of suspension obtained in 3.2 in order to verify the binding specificity of the anti-HbF antibody used.
[0316] The labeled and nonlabeled samples and also the isotype control were incubated for 15 minutes in the dark and at ambient temperature. The RBCs were subsequently washed in 2 ml of 1×PBS buffer (reagent D Fetal Cell Count™ kit, Cat IQP-349), followed by centrifugation at 300 G for 3 minutes at ambient temperature. The washed blood cell pellets while resuspended by adding 500 μl of 1×PBS buffer (reagent D Fetal Cell Count™ kit, Cat IQP-349). The red blood cells were analyzed in the cytometer within minutes following their pre-processing.
[0317] 3.4. Cytometer Sample Analysis
[0318] During the analysis, the samples were placed in a cold (ice) chamber and in the dark in order to prevent fluorescence losses. Before the data acquisition, the cytometer is precalibrated according to the supplier's recommendations.
[0319] The nonlabeled sample was used to determine the voltages (or PMT) of each parameter: FSC (Forward Scatter), SSC (Side Scatter) and fluorescence (table 1). The selection of the red blood cell populations was based on the criteria of size or FSC and granulosity or SSC on the Log FSC-A vs Log SSC-A cytogram while eliminating the debris and background noises. The negative control was determined on the signal generated by the sample not labeled by fluorescence (
TABLE-US-00001 TABLE 1 Adjustment of the cytometer obtained with the sample of nonlabeled red blood cells Parameter Voltage Log A H W FSC 341 ✓ ✓ ✓ ✓ SSC 328 ✓ ✓ ✓ ✓ PE 400 ✓ ✓ ✓ ✓
[0320] The exclusion of the red blood cell (RBC) doublets was carried out with a low flow rate by selecting the RBC populations on the FSC-W vs FSC-H then SSC-W vs FSC-H cytograms according to
[0321] A minimum number of 100 000 RBC events were collected in order to improve the accuracy of the analysis and to register a sufficient number of RBCs after the exclusion of the doublets. For each sample, the statistics were generated by the acquisition software (Diva®) with the number of events recorded for each RBC population selected. The fluorescence intensity (MFI) is given as geometric mean and arithmetic mean.
[0322] 4. Determination of the Amount of Labeled HbF Antigen Concentration Per Red Blood Cell or AG/RBC
[0323] In order to determine the amount of labeled HbF per red blood cell, the fluorescence intensity obtained with the flow cytometer was converted into amount of labeled HbF per red blood cell (AG/RBC) by means of the calibrated beads contained in a phycoerythrin fluorescence quantification kit (Becton Dickinson QuantiBRITE PE, Cat: 340495, BD Biosciences, Le Pont De Claix, France). These beads are conjugated to phycoerythrin in such ways to provide 4 levels of fluorescence (Low, Medlow, MedHigh and High) corresponding to a number of phycoerythrin molecules per bead (
[0324] 5. Determination of the HbF Content (Pq) of Each RBC on the Basis of AG/RBC and the MCHbFCo
[0325] The data recorded by the acquisition software of the flow cytometer (Diva®) were processed on FlowJo V10 (Cat 130-099-429 Miltenyi Biotec, Paris, France). This is a program for analysis and visualization of the cytometry data exported in “flow cytometry standard” or fcs format. The FlowJo software made it possible to generate the values of each parameter (FSC, SSC, Fluorescence) for each event.
[0326] For each RBC sample collected for this study, the distribution of HbF labeled with the antibody is represented by a histogram associating the number of red blood cells and the Log of fluorescence.
[0327] The mean HbF content per red blood cell (MCHbFCo) of each sample of selected patients, obtained by the combination of HPLC and of MCHCo, was associated with the mean fluorescence converted into AG/RBC by virtue of the quantification beads.
[0328] In order to determine the HbF content (in pg) of each red blood cell, a standard curve was constructed on the basis of the MCHbFCo values and the AG/RBC values, thus giving a range of known MCHbFCo values associated with the fluorescence (
Example 2: Validation of the Method for Determining the HbF Content of Each Red Blood Cell
[0329] 1. Characteristics of the Included Patients
[0330] A cohort of 12 patients was selected in order to constitute a range of HbF content, the criterion for selecting these patients was the confirmation of a homogeneous distribution, Log-Normal of their HbF. The data from these patients were used to construct a standard curve (cf. example 1, point 5) serving to determine the HbF content of each red blood cell. Table 2 summarizes the demographic data and the genetic characteristics of these patients.
TABLE-US-00002 TABLE 2 Demographic and biological data of the included patients for constructing the standard curve Age at inclusion Genetic HbF MCHCo MCHbFCo MCV Patients (years) Sex characteristics (%) (pg) (pg) (μm.sup.3) 1 18 Male β-thalassemia 100 24.5 24.5 73 2 62 Male β-thalassemia 69.6 27.2 18.93 91 3 50 Male β-thalassemia 61.5 20.1 12.36 64 4 46 Male β-thalassemia 53.2 19.3 10.27 63 5 50 Female Ghanaian HPFH2 34.80 28 9.74 81 6 39 Female β-thalassemia 27.9 28 7.76 81 7 39 Male β-thalassemia 21 23.8 5 72 8 43 Female β-thalassemia 16.4 23.6 3.87 71 9 37 Male β-thalassemia 14.9 18.5 2.76 62 10 60 Female β-thalassemia 2.7 23.4 3.11 67 11 58 Male β-thalassemia 0.8 19.4 0.15 64 12 23 Male β-thalassemia 0.8 27.8 0.22 83
[0331] A homogeneous distribution of the red blood cell fluorescences and therefore of the HbF content was verified on the set of samples collected (
[0332] 2. Test for Reproducibility of the Various Measurements
[0333] A reproducibility study was carried out by testing the variabilities on the following measurements: [0334] MCHbFCo on fresh red blood cells and on red blood cells after a thawing series [0335] MFI values obtained with the QuantiBRITE PE beads and the RBCs [0336] AG/RBC after a thawing series
[0337] No significant difference was observed on the MCHbFCo values (pg) obtained on fresh red blood cells and after 12 rounds of thawing (n=13, p>0.9999) or on the AG/RBC values measured after 11 rounds of thawing (p=0.73). Likewise, the analysis of variability of the data obtained on the quantification beads revealed no significant difference (n=11, p=0.99).
[0338] 3. Method for Measuring the HbF Content in Each Red Blood Cell
[0339] The MCHbFCo range obtained in the patients having a homogeneous distribution of the HbF contents per red blood cell was associated with the fluorescence intensities and then with the amounts of antigens per red blood cell by means of the calibration beads. A standard curve (or regression straight line) was determined from the AG/RBC means obtained after 11 rounds of thawing and the MCHbFCo values of each patient (
[0340] A coefficient of correlation at 97.18% (r.sup.2=0.9444) was obtained between the MCHbFCo values and the means of the amounts of HbF antigen (labeled by the fluorescent antibody) per red blood cell, for the various patients. This standard curve makes it possible to determine the HbF content per RBC for a given fluorescence value. The accuracy of the measurements is given by a confidence interval at 95% determined from the “mean” standard curve, from the standard deviations between each regression and from the number of the regression model having served to calculate the mean.
Example 3: Study of the HbF Distribution During the Treatment with Hydroxyurea
[0341] 1. Patients
[0342] A monocentric, longitudinal, prospective study was carried out on a cohort of 29 adult sickle cell disease patients (age 18 years old) exhibiting an SS homozygotes mutation, beginning a treatment with hydroxyurea, regularly monitored at the major sickle cell syndrome reference Center at the Centre Hospitalier Universitaire Henri Mondor [Henri Mondor University Hospital Center] in Créteil. Patients having had an attack requiring hospitalization and/or a transfusion exchange program during the previous 3 months before inclusion and also patients who were pregnant or who were wanting to get pregnant were excluded from the study.
[0343] The patients exhibiting the eligibility criteria were monitored at various times before and during the treatment with hydroxyurea: D0 (before the beginning of the treatment with hydroxyurea), at 15 days, 1 month, 3 months, 4 months and 6 months or more after the setting up of the treatment with hydroxyurea (D15, M1, M3, M4 and ≥M6 respectively).
[0344] In accordance with the Declaration of Helsinki on the ethical principles applicable to medical research involving human beings (World Medical Association, text in force 2008, paragraph 24), all recruited patients were informed of the risks and benefits of this study and provided written consent before inclusion. In accordance with the legislation in force (Articles L.1121-3 and R.5121-13 of the French Public Health Code), the information relating to the patients was protected anonymously in order to ensure confidentiality throughout the duration of the study.
[0345] This research protocol was approved by the International Review Board Ile-de-France IV located at the Hospital Saint-Louis (IRB No. 00003835).
[0346] 2. Methods
[0347] During each visit to the center (D0, D15, M1, M3, M4 and ≥M6), approximately 16 ml of blood were taken and collected in 4 tubes containing EDTA.
[0348] 2.1. Pretreatment of the Blood Cell Samples
[0349] The samples were treated within 24 h of being taken. The treatment involves the separation of the red blood cells as a function of their density and the cryopreservation of the blood cell fractions and of the total or non-fractionated red blood cells.
[0350] 3. Analysis of the Blood Cell Samples
[0351] The various measurements carried out on each blood cell fraction or on the total RBCs were composed of: [0352] complete blood count or blood hemogram by an automated counter; [0353] measurement of the mean HbF percentage (% HbF) by HPLC; and [0354] flow cytometry measurement of the fluorescence intensity of each RBC labeled with an antibody directed against HbF in accordance with the protocol detailed in example 1.
[0355] 3.1. Complete Blood Count, Measurement of the Mean HbF Percentage (% HbF) by HPLC
[0356] These analyses were carried out on fresh RBCs and RBCs after thawing according to the same techniques as those described in example 1.
[0357] 3.2. Measurement of the HbF Content of Each RBC by Flow Cytometry
[0358] This analysis was carried out after thawing of the RBCs stored at −80° C. according to the technique described in example 1.
[0359] The HbF content of each RBC was determined using the standard curve described in example 1 based on the fluorescence intensities of each red blood cell, converted into AG/RBC, using the QuantiBRITE PE beads. The measurements were carried out at each stage of the treatment (D0, D15, M1, M3, M4 and ≥M6) in the various blood cell fractions: non-dense, dense and total red blood cells.
[0360] 4. Statistical Analyses
[0361] In the SS patients taking hydroxyurea, the percentages of RBCs containing HbF contents grouped together in arbitrarily defined categories (0 to 2 pg, 2 to 4 pg, 4 to 6 pg, etc.) were compared during the longitudinal monitoring in order to determine the variations in the HbF distribution before and during the treatment with hydroxyurea. The comparisons between each stage of the treatment were carried out by means of an ANOVA measurement repeated between D0, D15-M3 and M6 in the total red blood cells.
[0362] The statistical analyses were carried out using the GraphPad Prism®Version 6 software (RITME Informatique Paris France).
[0363] 5. Results
[0364] 5.1. Demographic Data and Indications of Hydroxyurea
[0365] In total, 29 adult SS patients were included. Out of the entire cohort, 10 patients were monitored between D0 and ≥M6, the RBC samples of a patient were hemolyzed, 9 patients were analyzed.
[0366] The demographic data of the included patients are summarized in table 3
TABLE-US-00003 TABLE 3 Demographic data of SS patients taking hydroxyurea Men Women N 13 16 Age 33 [28.50; 45] 36 [31; 44.25] Age expressed as Median [25th percentile; 75th percentile]
[0367] The hydroxyurea was administered with an average dose of 15 mg/Kg/day for the following indications: VOC prevention, chronic visceral attacks, leg ulcers, sickle cell nephropathy, significant hemolysis with anemia, priapism, stroke
[0368] 5.2. Biological Data
[0369] The mean HbF percentage (% HbF) increases by approximately 3-fold after 6 months of treatment with hydroxyurea. The data are presented in table 4.
TABLE-US-00004 TABLE 4 Variation in %/HbF during the treatment with hydroxyurea p (D0 and D0 D15-M1 M3-M4 M6 M6) Total red 2.65 4.9 7 10.05 0.03 blood cells [1.8-6.35] [1.85-8.4] [2.4-17.80] [3.33-19.13] Data expressed as Median [25th percentile; 75th percentile]
[0370] A significant increase in the mean corpuscular volume (MCV) and in the mean hemoglobin content per red blood cell (MCHCo) was observed after 6 months of treatment with hydroxyurea, indicating good patient conformity and the efficacy of the treatment despite the low patient numbers. The percentage of dense red blood cells has a tendency to decrease; however, this decrease is not significant. Likewise, for the mean corpuscular hemoglobin concentration (MCHC), the leukocytes and the platelets, no significant variation was observed. The results are presented in table 5.
TABLE-US-00005 TABLE 5 Biological data before and during the treatment with hydroxyurea D0 M6 p Hemoglobin 8 9.35 0.2 (g/dl) [6.65-9.05] [7.30-10.10] Leukocytes 10.70 9 0.11 (Giga/l) [9.45-12.20] [5.35-11.58] MCV 87 100.50 0.03 (femtoliter) [83-95] [87.75-115] MCHC (g/dl) 32.60 34.40 0.08 [32.05-35.10] [32.43-35.70] MCHCo (pg) 29.70 33.80 0.008 [25.30-32.85] [30.70-39.65] Platelets 359 318 0.18 (giga/1) [288-548] [257-438.80] Data expressed as Median [25th percentile; 75th percentile]
[0371] 5.3. HbF Distribution During the Treatment with Hydroxyurea
[0372] The qualitative analysis by flow cytometry of the response to treatment with hydroxyurea shows a heterogeneous HbF distribution before the beginning of the treatment in 8 patients. After 6 months of treatment with hydroxyurea, the HbF follows a homogeneous distribution in 6 patients. The HbF distribution becomes homogeneous starting from M2 in 1 patient and after ≥M4 in 5 patients.
[0373] The HbF distribution was quantitatively analyzed by means of the method described in study 1. The number and percentage of red blood cells per HbF content range (pg) was determined for each patient. The analysis of the red blood cell distribution over time while taking hydroxyurea was carried out in 9 patients. The results show a significant variation in the red blood cells having the lowest HbF contents (0 and 2 pg) with an increase of approximately 25% between D0 and M6. The red blood cells which have an HbF content of greater than 20 g increase significantly, by up to 3 times, after 6 months of treatment with hydroxyurea; however, this variation is not significant. The red blood cells which have medium-high contents exhibit much lower variations.
TABLE-US-00006 TABLE 6 Red blood cell distribution per HbF content range under hydroxyurea treatment HbF D0 D15-M3 ≥M6 (pg) (% RBCs) (% RBCs) (% RBCs) p [0; 2[ 71.13 (23.16 65.63 (25.13 53.17 (30.63 0.0083 [2; 4[ 9.14 (7.44 11.32 (6.80 15.05 (10.11 0.1868 [4; 6[ 4.54 (3.86 4.88 (2.90 6.70 (4.39 0.2226 [6; 8[ 3.16 (2.92 3.42 (2.52 3.92 (2.44 0.5186 [8; 10[ 2.42 (2.32 2.65 (2.18 2.72 (1.83 0.6932 [10; 15[ 1.94 (1.84 2.41 (2.73 2.55 (2.57 0.3926 [15; 20[ 3.66 (3.35 4.42 (4.27 4.29 (3.42 0.474 ≥20 4.01 (4.35 5.27 (8.56 11.60 (18.64 0.1626 The data are expressed as mean (standard deviation)
[0374] The measurement of the fluorescence intensities of each red blood cell by flow cytometry made it possible to categorize each red blood cell as a function of the HbF content thereof. The analysis of the red blood cell distribution as a function of the HbF content thereof makes it possible to identify various treatment response profiles. By way of example, patients 11 and 24 (tables 7 and 8), expressing identical % HbF values at D0 (6%) and M6 (18%) with an MCHCo at D0=33.6 and 34.5 pg and at M6=38.9 and 42 pg (patients 11 and 24 respectively), exhibit 2 different distributions. In patient 11, a lower number of red blood cells is noted with an HbF content of less than 2 pg with a decrease of approximately 73% at M6, whereas patient 24 exhibits a higher number of red blood cells with an HbF content of less than 2 pg with a decrease of approximately 40% at M6. The HbF distribution in patient 11 is described by a bimodal curve at D0 and at M6, whereas the distribution of patient 24 exhibits a relatively homogeneous distribution over the low HbF amounts with a clear shift of the set toward higher amounts.
TABLE-US-00007 Patient 11 HbF D0 D15-M1 M3-M4 ≥M6 (pg) (% RBCs) (% RBCs) (% RBCs) (% RBCs) [0; 2[ 48.44 55.38 48.83 12.93 [2; 4[ 26.27 23.31 31.73 17.62 [4; 6[ 8.13 6.25 9.30 11.85 [6; 8[ 3.77 3.07 4.32 8.05 [8; 10[ 2.69 2.55 2.04 6.08 [10; 15[ 2.41 2.24 0.72 6.13 [15; 20[ 4.21 4.42 2.46 9.77 ≥20 4.08 2.78 0.59 27.57
TABLE-US-00008 Patient 24 HbF D0 D15-M1 M3-M4 ≥M6 (pg) (% RBCs) (% RBCs) (% RBCs) (% RBCs) [0; 2[ 60.82 53.17 44.56 36.98 [2; 4[ 9.38 11.57 24.93 25.28 [4; 6[ 4.83 6.69 12.53 13.64 [6; 8[ 4.01 5.58 5.81 6.92 [8; 10[ 3.63 4.50 3.09 3.93 [10; 15[ 3.83 3.89 1.87 2.70 [15; 20[ 6.38 7.56 3.81 5.36 ≥20 7.12 7.04 3.41 5.20
[0375] Tables 7 and 8: distribution of each red blood cell as a function of the content thereof of HbF categories (compared with total red blood cells) before and during the treatment with hydroxyurea.
[0376] The biological data of patients 11 and 24 were also analyzed (table 9 below).
TABLE-US-00009 TABLE 9 Biological data of patients 11 and 24. HbF = Hemoglobin F; RETIC = Reticulocytes; LDH = Lactate dehydrogenase; ASAT = Aspartate transaminase Relative Relative difference in difference in Patient 11 Patient 11 Patient 24 Patient 24 % D 0 vs M 6 % D 0 vs M 6 (D 0) (M 6) (D 0) (M 6) (patient 11) (patient 24) HbF (g/dl) 6.2 7.1 7.6 8.7 +14.5 +14.5 RETIC (g/l 275 63 271 116 −77.1 −57.2 LDH (IU/l) 905 383 509 303 −57.7 −40.5 ASAT (IU/l) 55 26 32 29 −52.7 −9.4
[0377] The biological data showed that patients 11 and 24 exhibit identical relative differences in HbF (+14.5%) before and after six months of treatment with hydroxyurea. They nevertheless exhibit considerable differences in terms of HbF distribution (see table 8). Patient 11, compared with patient 24, exhibits, before treatment (D0), fewer RBCs having high protective HbF contents (17% RBCs vs 25% RBCs>6 pg of HbF), which might explain a greater anemia (6.2 g/dl vs 7.6 g/dl) and a greater hemolysis (LDH at 905 IU/I vs 509 IU/I). On the other hand, the increase in RBCs>6 pg HbF in patient 11 was more marked than in patient 24, at 6 months of treatment (57% vs 24%), and might explain a greater decrease in hemolysis under treatment (RETIC: −77% vs −57%; LDH: −57% vs −40.5%; ASAT −52% vs −9.4%).
[0378] Another example of distribution as a function of the HbF content of each red blood cell use represented for patient 2. Patient 2 exhibited, before the treatment with hydroxyurea, a relative homogeneity of HbF content distribution. However, at M6, the distribution was characterized by a clear increase in red blood cells containing a content greater than 20 pg to the detriment of all the other categories (table 10). It should be noted that the % HbF varies from 6% to 25% between D0 and M6, which is similar to the 2 previous examples.
TABLE-US-00010 TABLE 10 distribution of red blood cells as a function of the content thereof of HbF categories (compared with total red blood cells) before and during the treatment with hydroxyurea for patient 2 D0 D15-M1 M3-M4 ≥M6 HbF (pg) (% RBCs) (% RBCs) (% RBCs) (% RBCs) [0; 2[ 22.43 9.21 5.29 4.98 [2; 4[ 15.76 14.72 8.62 7.04 [4; 6[ 12.85 9.52 10.61 5.53 [6; 8[ 10.04 8.47 10.39 4.85 [8; 10[ 7.84 7.35 8.75 4.56 [10; 15[ 5.79 9.03 8.50 7.48 [15; 20[ 11.05 14.27 14.54 9.75 ≥20 14.24 27.43 33.30 55.81
[0379] 6. Conclusion on the HbF Determination
[0380] The data showed that the red blood cells having a very low HbF content decreased greatly and that the red blood cells having an HbF content greater than 20 pg increase considerably, up to 3-fold, after 6 months of treatment with hydroxyurea.
[0381] 7. Determination of the HbS Content (Pg) of Each RBC on the Basis of AG/RBC and the MCHbSCo
[0382] The HbS/RBC was quantified in an unknown sample from SCD patients (not included in the linear regression,
[0383] With the exception of the used anti-hemoglobin antibody (Anti HbS (mouse) Monoclonal Antibody 200 301 GS5, ROCKLAND® Number: 200 301 GS5.) Monoclonal Antibody), the same materials and methods were applied as described in the example “Determination of the HbF content (pg) of each RBC on the basis of AG/RBC and the MCHbFCo” above.
[0384] The FACS method allowed the HbS quantification in an unknown blood sample from a first subject (
TABLE-US-00011 Subject 1: Mean HbS amount (HPLC): 18.5 HbS (pg) RBC % 0-5 0.1 5-10 1.0 10-15 6.0 15-20 13.9 20-25 18.1 25-30 18.1 30-35 15.7 35-40 12.3 40-45 8.7 45-50 6.1 Subject 2: Mean HbS amount (HPLC): 29 HbS (pg) RBC % 0-5 0.0 5-10 1.4 10-15 4.7 15-20 8.8 20-25 13.7 25-30 17.4 30-35 17.0 35-40 15.0 40-45 12.2 45-50 9.8
[0385] Table 11 showing the results of the HbS determination according to the invention in subject 1 and subject 2.
[0386] As per in the case of the HbF determination, the present method allowed a detailed analysis of the HbS content in each cell of the blood sample.
[0387] 7. Determination of the HbA Content (Pq) of Each RBC on the Basis of AG/RBC and the MCHbFCo
[0388] The HbA/RBC was quantified in an unknown sample from two SCD patients (not included in the linear regression,
[0389] With the exception of the used anti-hemoglobin antibody (Anti HbA (mouse) Monoclonal Antibody 200 301 GS4, ROCKLAND® Number: 200 301 GS4), the same materials and methods were applied as described in the example “Determination of the HbF content (pg) of each RBC on the basis of AG/RBC and the MCHbFCo” above.