Use of VDAC and lamp1 as markers for detecting pocked red blood cells

Abstract

OF THE INVENTION USE OF VDAC AND LAMP1 AS MARKERS FOR DETECTING POCKED RED BLOOD CELLS Counting vacuole-containing RBC (pocked-RBC) is a robust marker of defective spleen function but requires the expertise of fewer and fewer specialized laboratory technicians. Now the inventors have identified VDAC and LAMP1 as markers for detecting pocked red blood cells. In particular, after performing a proteomic analysis, the inventors confirmed the presence of these proteins in splenectomized subjects by western blot. RBC of splectomized and control subjects were then marked with antibodies LAMP1 or VDAC and passed to cytometry. RBC from control subjects did not have labelling but there were RBC marked for splenectomized subjects. RBC labelling were then observed in DIC and pocked cells with vacuoles marked were quantified. No marked vacuole was observed in control subjects whereas between 4 and 33% of pocked cells had at least one vacuole marked with antibodies LAMP1 or VDAC. Since, these proteins are not found in control subjects, it is therefore possible to use this markers for detecting pocked RBCs and thus splenectomized subjects.

Description

[0001] USE OF VDAC AND LAMP1 AS MARKERS FOR DETECTING POCKED RED BLOOD CELLS

[0002] FIELD OF THE INVENTION:

[0003] The present invention is in the field of medicine, in particular haematology.

[0004] BACKGROUND OF THE INVENTION:

[0005] The spleen protects against blood-borne infections, contributes to the maturation of reticulocytes1, and filters red blood cells (RBC)2'3. Defective spleen function (hyposplenism) is caused by splenectomy or by immunological or hematological diseases4-6. It occurs very soon in Sickle Cell Disease (SCD)7, without clear correlation with spleen size8. Spleen function is assessed by scintigraphy, and by quantifying circulating blood cells such as marginal zone B lymphocytes, RBC containing Howell-Jolly Bodies (HJB), or vacuoles-containing RBC (pocked-RBC). These RBC subpopulations are cleared of their inclusions as they pass through the spleen in a process called pitting9-11. Scintigraphy uses radiolabeled items12,13and is semi- quantitative14. Numbers of circulating IgM memory B cells are low in hyposplenism, but no threshold robustly discriminates hyposplenic from healthy subjects15. Despite recent improvements16,17, counting HJB lacks specificity and sensitivity, likely because of their relative small number in circulation (<2%) in most asplenic subjects8. Circulating pocked-RBC, visualized using differential interference contrast (DIC) microscopy, are >4.5% in hyposplenic patients18, above 10% in Jamaican children with SCD19, and from 20-50% in splenectomized subjects9,10. Pocked-RBC counts correlate with spleen intensity on scintigraphy20and display a wide window of quantification, but this method requires an expert microscopist, is timeconsuming and poorly reproducible from site to site.

[0006] SUMMARY OF THE INVENTION:

[0007] The present invention is defined by the claims. In particular, the present invention relates to methods for detecting pocked red blood cells and use thereof for assessing the spleen function of a subject.

[0008] DETAILED DESCRIPTION OF THE INVENTION: The present invention relates to a method for detecting pocked red blood cells in a sample comprising the steps consisting of contacting the sample with a panel of binding partners specific for markers that include LAMP1 and VDAC wherein pocked red blood cells express said VDAC and / or LAMP1.

[0009] As used herein, the term “red blood cells” or “RBCs” has its general meaning in the art and refers to highly-specialized cells responsible for delivery of oxygen to, and removal of carbon dioxide from, metabolically-active cells via the capillary network. They are shaped as biconcave discs and average about 8-10 microns in diameter. Early erythroid cells are nucleated and express CD71 and CD235a on their surface; as cells mature and migrate from the bone marrow to the periphery, they lose their nucleus and become reticulocytes, still containing ribosomal RNA, which is then lost roughly together with CD71 expression. The absence of CD45 - which is otherwise present on all differentiated hematopoietic cells - can be used as a negative marker for red blood cells (Samsel, L., & McCoy, J. P., Jr. (2015). Imaging flow cytometry for the study of erythroid cell biology and pathology. Journal of immunological methods. 423, 52 59).

[0010] Thus, in some embodiments, the panel of binding partners comprises one or more binding partner(s) specific for a phenotypic marker of red blood cells selected from the group consisting of CD173, CD175, CD234, CD235a, CD235b, CD236, CD238, CD239, CD240CE, CD240D, CD241, CD242, CD297, CD45, CD55, CD59, and CD71.

[0011] As used herein, the term “pocked red blood cells” or “pocked RBCs” has its general meaning in the art and refers to vacuoles-containing RBC as defined in Holroyde CP, Oski FA, Gardner FH. The "pocked" erythrocyte. Red-cell surface alterations in reticuloendothelial immaturity of the neonate. N Engl J Med. 1969 Sep 4;281(10):516-20. doi: 10.1056 / NEJM196909042811002. PMID: 4895379. The term is also known as pitted or vacuolated RBCs.

[0012] As used herein, the term “VDAC” has its general meaning in the art and refers to a P-barrel membrane protein located in the outer mitochondrial membrane (OMM). VDAC conductance states play major roles in regulating permeability of ATP / ADP, regulation of calcium homeostasis, calcium flux within ER-mitochondria contact sites, and apoptotic signaling events. In particular, the term “VDAC” refers to the VDAC isoforms that include VDAC1, VDAC2 and VDAC3. Human VDAC1 set forth in SEQ ID NO: 1 is a 283 amino acid protein; human VDAC2 set forth in SEQ ID NO:2 is a 294 amino acid protein; and human VDAC3 set forth in SEQ ID NO:3 is a 283 amino acid protein.

[0013] SEQ ID NO : 1 >sp | P21796 | VDAC1 HUMAN Voltage-dependent anion-selective channel protein 1 OS=Homo sapiens OX=9606 GN=VDAC1 PE=1 SV=2 MAVPPTYADLGKSARDVFTKGYGFGLIKLDLKTKSENGLEFTSSGSANTETTKVTGSLET KYRWTEYGLTFTEKWNTDNTLGTEITVEDQLARGLKLTFDSSFSPNTGKKNAKIKTGYKR EHINLGCDMDFDIAGPSIRGALVLGYEGWLAGYQMNFETAKSRVTQSNFAVGYKTDEFQL HTNVNDGTEFGGSIYQKVNKKLETAVNLAWTAGNSNTRFGIAAKYQIDPDACFSAKVNNS SLIGLGYTQTLKPGIKLTLSALLDGKNVNAGGHKLGLGLEFQA

[0014] SEQ ID NO : 2 >sp | P45880 | VDAC2 HUMAN Voltage-dependent anion-selective channel protein 2 OS=Homo sapiens OX=9606 GN=VDAC2 PE=1 SV=2 MATHGQTCARPMCI PPSYADLGKAARDI FNKGFGFGLVKLDVKTKSCSGVEFSTSGSSNT DTGKVTGTLETKYKWCEYGLTFTEKWNTDNTLGTEIAIEDQICQGLKLTFDTTFSPNTGK KSGKIKSSYKRECINLGCDVDFDFAGPAIHGSAVFGYEGWLAGYQMTFDSAKSKLTRNNF AVGYRTGDFQLHTNVNDGTEFGGSIYQKVCEDLDTSVNLAWTSGTNCTRFGIAAKYQLDP TASI SAKVNNSSLIGVGYTQTLRPGVKLTLSALVDGKSINAGGHKVGLALELEA

[0015] SEQ ID NO : 3 >sp | Q9Y277 | VDAC3 HUMAN Voltage-dependent anion-selective channel protein 3 OS=Homo sapiens OX=9606 GN=VDAC3 PE=1 SV=1 MCNTPTYCDLGKAAKDVFNKGYGFGMVKIDLKTKSCSGVEFSTSGHAYTDTGKASGNLET KYKVCNYGLTFTQKWNTDNTLGTEI SWENKLAEGLKLTLDTI FVPNTGKKSGKLKASYKR DCFSVGSNVDIDFSGPTIYGWAVLAFEGWLAGYQMSFDTAKSKLSQNNFALGYKAADFQL HTHVNDGTEFGGSIYQKVNEKIETSINLAWTAGSNNTRFGIAAKYMLDCRTSLSAKVNNA SLIGLGYTQTLRPGVKLTLSALIDGKNFSAGGHKVGLGFELEA

[0016] Thus in some embodiments, the panel of binding partners comprises either i) at least one binding partner specific for VDAC1, VDAC2 or VDAC3 or ii) an only one binding partner capable of binding to all VDAC isoforms (i.e. pan-isoform binding partner).

[0017] As used herein, the term “LAMP1” has its general meaning in the art and refers to the lysosome-associated membrane glycoprotein 1. The term is also known as CD107a. LAMP1 resides primarily across lysosomal membranes, and functions to provide selectins with carbohydrate ligands. Human LAMP1 set forth in SEQ ID NO:4 is a 417 amino acid protein.

[0018] SEQ ID NO : 4 >sp | Pl 1279 | LAMP1 HUMAN Lysosome-as sociated membrane glycoprotein 1 OS=Homo sapiens OX=9606 GN=LAMP1 PE=1 SV=3 MAAPGSARRPLLLLLLLLLLGLMHCASAAMFMVKNGNGTACIMANFSAAFSVNYDTKSGP KNMTFDLPSDATWLNRSSCGKENTSDPSLVIAFGRGHTLTLNFTRNATRYSVQLMSFVY NLSDTHLFPNASSKEIKTVESITDIRADIDKKYRCVSGTQVHMNNVTVTLHDATIQAYLS NSSFSRGETRCEQDRPSPTTAPPAPPSPSPSPVPKSPSVDKYNVSGTNGTCLLASMGLQL NLTYERKDNTTVTRLLNINPNKTSASGSCGAHLVTLELHSEGTTVLLFQFGMNASSSRFF LQGIQLNTILPDARDPAFKAANGSLRALQATVGNSYKCNAEEHVRVTKAFSVNI FKVWVQ AFKVEGGQFGSVEECLLDENSMLI PIAVGGALAGLVLIVLIAYLVGRKRSHAGYQTI As used herein, the term “expression” may refer alternatively to the transcription of a molecule (i.e. expression of the mRNA) or to the translation (i.e. expression of the protein) of a molecule. In some embodiments, detecting the expression may correspond to an intracellular detection. In some embodiments, detecting the expression may correspond to a surface detection, i.e. to the detection of molecule expressed at the cell surface. In some embodiments, detecting the expression may correspond to an extracellular detection, i.e. to the detection of secretion. In some embodiments, detecting the expression may correspond to intracellular, surface and / or extracellular detections. As used herein, the terms “expressing or “+” and “not expressing” or are well known in the art and refer to the expression level of the phenotypic marker of interest, in that the expression level of the phenotypic marker corresponding to “+” is high or intermediate, also referred as The phenotypic marker corresponding to is a null expression level of the phenotypic marker or also refers to less than 10 % of a cell population expressing the said phenotypic marker.

[0019] As used herein, the term “detecting” refers broadly to any means of determining the presence or absence of an object (e.g. pocked red blood cells). Thus “detecting” may include determining, assessing or measuring in any way or form whether or not the object is present — it may include qualitative, quantitative or semi -quantitative determinations.

[0020] As used herein, term “sample” whole blood sample, red blood cell concentrates and any other sample that contains red blood cells.

[0021] In some embodiments, the binding partners include but are not limited to antibodies, aptamer, and peptides. The binding partners will allow for the screening of cellular populations expressing the marker. Various techniques can be utilized to screen for cellular populations expressing the markers of interest, and typically include magnetic separation using antibody- coated magnetic beads, “panning” with antibody attached to a solid matrix (i.e., plate), and flow cytometry (See, e.g., U.S. Pat. No. 5,985,660; and Morrison et al. Cell, 96:737-49 (1999)).

[0022] In some embodiments, the binding partners are antibodies that may be polyclonal or monoclonal, preferably monoclonal, specifically directed against one cell surface marker. Polyclonal antibodies of the invention or a fragment thereof can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others. Various adjuvants known in the art can be used to enhance antibody production. Although antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred. Monoclonal antibodies of the invention or a fragment thereof can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique originally; the human B-cell hybridoma technique; and the EBV-hybridoma technique.

[0023] Typically, the binding partners are conjugated with a label for use in separation. Labels include magnetic beads, which allow for direct separation, biotin, which can be removed with avidin or streptavidin bound to a support, fluorochromes, which can be used with a fluorescence activated cell sorter, or the like, to allow for ease of separation of the particular cell type. Fluorochromes that find use include phycobiliproteins, e.g. phycoerythrin and allophycocyanins, fluorescein and Texas red. Typically each antibody is labeled with a different fluorochrome, to permit independent sorting for each marker. Suitable fluorescent detection elements include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade Blue™, Texas Red, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LC Red 705 and Oregon green. Suitable optical dyes are described in the 1996 Molecular Probes Handbook by Richard P. Haugland, hereby expressly incorporated by reference. Suitable fluorescent labels also include, but are not limited to, green fluorescent protein (GFP; Chalfie, et al., Science 263(5148):802-805 (Feb. 11, 1994); and EGFP; Clontech — Genbank Accession Number U55762), blue fluorescent protein (BFP; 1. Quantum Biotechnologies, Inc. 1801 de Maisonneuve Blvd. West, 8th Floor, Montreal (Quebec) Canada H3H 1J9; 2. Stauber, R. H. Biotechniques 24(3):462-471 (1998); 3. Heim, R. and Tsien, R. Y. Curr. Biol. 6:178-182 (1996)), enhanced yellow fluorescent protein (EYFP; 1. Clontech Laboratories, Inc., 1020 East Meadow Circle, Palo Alto, Calif. 94303), luciferase (Ichiki, et al., J. Immunol. 150(12):5408- 5417 (1993)), (P-galactosidase (Nolan, et al., Proc Natl Acad Sci USA 85(8):2603-2607 (April 1988)) and Renilla WO 92 / 15673; WO 95 / 07463; WO 98 / 14605; WO 98 / 26277; WO 99 / 49019; U.S. Pat. Nos. 5,292,658; 5,418,155; 5,683,888; 5,741,668; 5,777,079; 5,804,387; 5,874,304; 5,876,995; and 5,925,558). All of the above-cited references are expressly incorporated herein by reference. In some embodiments, detection elements for use in the present invention include: Alexa-Fluor dyes (an exemplary list including Alexa Fluor® 350, Alexa Fluor® 405, Alexa Fluor® 430, Alexa Fluor® 488, Alexa Fluor® 500, Alexa Fluor® 514, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 610, AlexaFluor® 633, Alexa Fluor® 647, Alexa Fluor® 660, Alexa Fluor® 680, Alexa Fluor® 700, and Alexa Fluor® 750), Cascade Blue, Cascade Yellow and R- phycoerythrin (PE) (Molecular Probes) (Eugene, Oreg.), FITC, Rhodamine, and Texas Red (Pierce, Rockford, Ill.), Cy5, Cy5.5, Cy7 (Amersham Life Science, Pittsburgh, Pa.). Tandem conjugate protocols for Cy5PE, Cy5.5PE, Cy7PE, Cy5.5APC, Cy7APC are known in the art. Fluorophores bound to antibody or other binding element can be activated by a laser and reemit light of a different wavelength. The amount of light detected from the fluorophores is related to the number of binding element targets associated with the cell passing through the beam. Any specific set of detection elements, e.g. fluorescently tagged antibodies, in any embodiment can depend on the types of cells to be studied and the presence of the activatable element within those cells. Several detection elements, e.g. fluor ophore-conjugated antibodies, can be used simultaneously, so measurements made as one cell passes through the laser beam consist of scattered light intensities as well as light intensities from each of the fluorophores. Thus, the characterization of a single cell can consist of a set of measured light intensities that may be represented as a coordinate position in a multi-dimensional space. Considering only the light from the fluorophores, there is one coordinate axis corresponding to each of the detection elements, e.g. fluorescently tagged antibodies. The number of coordinate axes (the dimension of the space) is the number of fluorophores used. Modem flow cytometers can measure several colors associated with different fluorophores and thousands of cells per second. Thus, the data from one subject can be described by a collection of measurements related to the number of antigens for each of (typically) many thousands of individual cells. See Krutzik et al., High- content single-cell drug screening with phosphospecific flow cytometry. Nature Chemical Biology, Vol. 4 No. 2, Pgs. 132-42, February 2008. Such methods may optionally include the use of barcoding to increase throughput and reduce consumable consumption. See Krutzik, P. and Nolan, G., Fluorescent cell barcoding in flow cytometry allows high-throughput drug screening and signaling profiling. Nature Methods, Vol. 3 No. 5, Pgs. 361-68, May 2006.

[0024] In some embodiments, the detection of the pocked red blood cells is carried out by a flow cytometric method.

[0025] As used herein, the term "flow cytometric method" refers to a technique for counting cells of interest, by suspending them in a stream of fluid and passing them through an electronic detection apparatus. Flow cytometric methods allow simultaneous multiparametric analysis of the physical and / or chemical parameters of up to thousands of events per second, such as fluorescent parameters. Modern flow cytometric instruments usually have multiple lasers and fluorescence detectors. A common variation of flow cytometric techniques is to physically sort particles based on their properties, so as to purify or detect populations of interest, using "fluorescence-activated cell sorting".

[0026] As used herein, "fluorescence-activated cell sorting" or “FACS” refers to a flow cytometric method for sorting a heterogeneous mixture of cells from a biological sample into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell and provides fast, objective and quantitative recording of fluorescent signals from individual cells as well as physical separation of cells of particular interest. Accordingly, FACS can be used with the methods described herein to isolate and detect the population of cells of the present invention. For example, fluorescence activated cell sorting (FACS) may be therefore used and typically involves a flow cytometer capable of simultaneous excitation and detection of multiple fluorophores, such as a BD Biosciences FACSSymphony™ flow cytometer, used substantially according to the manufacturer's instructions. The cytometric systems may include a cytometric sample fluidic subsystem, as described below. In addition, the cytometric systems include a cytometer fluidically coupled to the cytometric sample fluidic subsystem. Systems of the present disclosure may include a number of additional components, such as data output devices, e.g., monitors, printers, and / or speakers, softwares (e.g. (Flowjo, Laluza.... ), data input devices, e.g., interface ports, a mouse, a keyboard, etc., fluid handling components, power sources, etc. More particularly, the blood sample is contacted with a panel of antibodies specific for the specific market of the population of cells of the interest.

[0027] In some embodiments the binding partner is conjugated to a metallic chemical element such as lanthanides. Lanthanides offer several advantages over other labels in that they are stable isotopes, there are a large number of them available, up to 100 or more distinct labels, they are relatively stable, and they are highly detectable and easily resolved between detection channels when detected using mass spectrometry. Lanthanide labels also offer a wide dynamic range of detection. Lanthanides exhibit high sensitivity, are insensitive to light and time, and are therefore very flexible and robust and can be utilized in numerous different settings. Lanthanides are a series of fifteen metallic chemical elements with atomic numbers 57-71. They are also referred to as rare earth elements. Lanthanides may be detected using CyTOF technology. CyTOF is inductively coupled plasma time-of-flight mass spectrometry (ICP-MS). CyTOF instruments are capable of analysing up to 1000 cells per second for as many parameters as there are available stable isotope tags.

[0028] Since VDAC and LAMP1 are intracellular markers, the method of the present can typically involve the permeabilization of the cells preliminary to flow cytometry. Any convenient means of permeabilizing cells may be used in practicing the methods.

[0029] Typically, the binding partners are added to a suspension of cells, and incubated for a period of time sufficient to bind the available markers of interest (i.e. VDAC and LAMP1). The incubation will usually be at least about 5 minutes and usually less than about 30 minutes. It is desirable to have a sufficient concentration of binding partners in the reaction mixture, such that the efficiency of the separation is not limited by lack of binding partners. The appropriate concentration is determined by titration. The medium in which the cells are separated will be any medium that maintains the viability of the cells.

[0030] The method of the present invention is particularly suitable for assessing the spleen function of a subject, since the pocked red blood cells counts is correlated with the spleen function. Many diseases are associated with a dysfunctional spleen including congenital disorders (Congenital asplenia (isolated), Ivemark’s syndrome, Stormorken’s syndrome, Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) syndrome, Fetal hydantoin syndrome, Congenital cyanotic heart disease), sickle haemoglobinopathies (e.g. thalassemia and sickle cell disease), gastrointestinal diseases, hepatic disorders, autoimmune disorders, haematological / neoplastic disorders, sepsis and infectious diseases, and splenic diseases (e.g. splenic artery thrombosis, splenic vein thrombosis. . .). In particular, the method of the present invention is of a particular interest for patients suffering from haematological conditions such as P-hemoglobinopathies such as sickle cell disease or thalassemia (e.g. P-thalassemia). For patients suspected to have a spleen with diminished function, it is important to quantify their spleen function in order to assess the risk of developing overwhelming post-splenectomy infection. Subsequently, preventive measurements can be taken and, in the case of infection, therapy can be started without delay.

[0031] Accordingly, a further object of the present invention relates to a method of assessing the spleen function of a subject comprising the step of detecting the pocked blood cells present in a sample obtained from the subject by implemented the method as above described wherein the pocked red blood cells count correlates with the spleen function of the subject.

[0032] Typically, the higher is the pocked red blood cell count, the worse is the spleen function of the subject.

[0033] In some embodiments, the method comprises the steps of i) detecting the pocked red blood cells in the sample obtained from the patient, ii) comparing the count determined at step i) with a predetermined reference value, iii) and concluding that the patient has a good spleen function when the count quantified at step i) is lower than the predetermined reference value or concluding that the patient has a poor spleen function when the count determined at step i) is higher than the predetermined reference value.

[0034] Typically, the predetermined reference value is a threshold value or a cut-off value. 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 recognized by a person of ordinary skilled in the art. For example, retrospective quantification of pocked red blood cells in properly banked historical subject samples may be used in establishing the predetermined 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. For example, after determining the pocked red blood cell count in a group of reference, one can use algorithmic analysis for the statistic treatment of the measured amounts of SMEs in samples to be tested, and thus obtain a classification standard having significance for sample classification. 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 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. Then sensitivity is used as the vertical coordinate and specificity is used as the horizontal coordinate to draw a curve. The higher the area under the curve (AUC), the higher the accuracy of diagnosis. On the ROC curve, the point closest to the far upper left of the coordinate diagram is a critical point having both high sensitivity and high specificity values. The AUC value of the ROC curve is between 1.0 and 0.5. When AUC>0.5, the diagnostic result gets better and better as AUC approaches 1. When AUC is between 0.5 and 0.7, the accuracy is low. When AUC is between 0.7 and 0.9, the accuracy is moderate. When AUC is higher than 0.9, the accuracy is quite high. This algorithmic method is preferably done with a computer. 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), etc. In some embodiments, a cut-off value consists of a range of quantification values, e.g. centered on the quantification value for which the highest statistical significance value is found. For example, on a hypothetical scale of 1 to 10, if the ideal cut-off value (the value with the highest statistical significance) is 5, a suitable (exemplary) range may be from 4-6. For example, a patient may be assessed by comparing values obtained by measuring the pocked red blood cell count, where values greater than 5 reveal that the patient has a poor spleen function and values less than 5 reveal that the patient has a good spleen function. In some embodiments, a patient may be assessed by comparing values obtained by measuring the pocked red blood cell count and comparing the values on a scale, where values above the range of 4-6 indicate that the patient is a poor spleen function and values below the range of 4-6 indicate that the patient has a good spleen function, with values falling within the range of 4-6 indicate that further explorations are needed to conclude whether the patient has or has not a good spleen function.

[0035] The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

[0036] FIGURES:

[0037] Figure 1: Western Blot. A. Western Blot realized on 6 splenectomized (SPL) and 5 control (CTL) subjects. Antibodies used: VDAC, LAMP1 and Actin ; B. Amount of proteins observed in WB on CTL and SPL subjects Figure 2: Cytometry. Quantification of RBC labelling in cytometry on 4 splenectomized subjects.

[0038] Figure 3: Immunofluorescence. Quantification of pocked cells with at least one vacuole marked

[0039] EXAMPLE:

[0040] Material & Methods:

[0041] Mature RBC sorting and proteomic (5 controls and 5 splenectomised subjects):

[0042] RBC were washed 3 times in PBS / Albumax 1% and pellet was incubated 20 minutes in Thiazole Orange Dye (diluted 1 / 3000 in PBS / Albumax 1%) at 0.1% hematocrit. Mature RBC were sorted (30 million), washed three times in PBS, divided in 3 tubes and frozen at -80°C. The first tube has been used for proteomic like “RBC mature”. The others tubes have been used to do ghost. For that, RBC were incubated in 5P8 during 20min and centrifugated 20 minutes at 15 000g a 4°C. Pellet was washed until the membranes are completely white. The second tube was frozen and used for proteomic as “ghost” at -80°C and send to the proteomic platform.

[0043] Western Blot:

[0044] The third was used to protein dosage with Pierce BCA Protein Assay Kit and the rest of the tube was used to western blot. After migration and transfer, membranes were saturated during Ih and incubated with VDAC or LAMP1 (diluted 1 / 1000) overnight. Then, membranes were washed three times and incubated with secondary antibody goat anti-rabbit (diluted 1 / 5000) during Ih. Membranes were revealed, washed 3 times and incubated with coupled actine during Ih and revealed another time.

[0045] VDAC / Lampl Labelling and cytometry:

[0046] RBC were fixed in PBS / Albumax 0.5% / PFA 2% / glutaraldehyde 0.0075% during 30 minutes and washed twice in PBS / Albumax 1%. Then, pellet was saturated and permeabilized in PBS / Albumax / Triton 0.1% during Ih and washed in PBS / Albumax 1%. After that, pellet was incubated over night with VDAC (diluted 1 / 150 in PBS) or LAMP1 (diluted 1 / 200 in PBS) and washed twice in PBS. To finish, pellet was incubated 2h with AF555 diluted 1 / 200 for VDAC labelling or 1 / 300 for LAMP1 labelling, washed 2 times in PBS and resuspended in 200pl PBS to observe with a cytometry.

[0047] Results

[0048] Matures RBC and ghosts of 5 splenectomized subjects (with >30% of pocked cells) and 5 control subjects (<5% of pocked cells) were studied by proteomic. 31 proteins were increased or appeared in matures RBC and ghosts of splenectomized subjects (data not shown). On these 31 proteins, 8 are vesicular, 3 play a role on reticulum endoplasmic and the 20 others are mitochondrial.

[0049] Based on the proteomic analysis, two proteins were chosen for cytometry and immunofluorescence characterizations: one lysosomal protein (LAMP1) and one mitochondrial protein (VDAC).

[0050] First, the presence of these proteins in splenectomized subjects was confirmed by western blot whereas these proteins were absent in control subjects (Figure 1).

[0051] RBC of splenectomized and control subjects were marked with antibodies LAMP1 or VDAC and passed to cytometry. RBC from control subjects did not have labelling but there were RBC marked for splenectomized subjects (Figure 2). RBC labelling were then observed in DIC and pocked cells with vacuoles marked were quantified. No marked vacuole was observed in control subjects whereas between 4 and 33% of pocked cells had at least one vacuole marked with antibodies LAMP1 or VDAC (Figure 3).

[0052] In conclusion, these results suggest that these proteins are presented in vacuoles of pocked cells of splenectomized subjects. These proteins are not found in control subjects, it is therefore possible to use these markers for detecting pocked RBCs and thus splenectomized subjects.

[0053] REFERENCES:

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Claims

CLAIMS:

1. A method for detecting pocked red blood cells in a sample comprising the steps consisting of contacting the sample with a panel of binding partners specific for markers that include LAMP1 and VDAC wherein pocked red blood cells express said VDAC and / or LAMP1.

2. The method of claim 1 wherein the panel of binding partners comprises one or more binding partner(s) specific for a phenotypic marker of red blood cells selected from the group consisting of CD173, CD175, CD234, CD235a, CD235b, CD236, CD238, CD239, CD240CE, CD240D, CD241, CD242, CD297, CD45, CD55, CD59, and CD71.

3. The method according to claim 1 or 2 wherein the panel of binding partners comprises either i) at least one binding partner specific for VDAC1, VDAC2 or VDAC3 or ii) an only one binding partner capable of binding to all VDAC isoforms (i.e. pan-isoform binding partner).

4. The method according to any one of claims 1 to 3 wherein the binding partners include antibodies, aptamer, or peptides5. The method of claim 4 wherein the binding partners are antibodies, preferably monoclonal antibodies.

6. The method according to any one of claims 1 to 5 wherein the detection of the pocked red blood cells is carried out by a flow cytometric method, preferably fluorescence- activated cell sorting.

7. A method of assessing the spleen function of a subject comprising the step of detecting the pocked blood cells present in a sample obtained from the subject by implemented the method according to any one of claims 1 to 6 wherein the pocked red blood cells count correlates with the spleen function of the subject.

8. The method of claim 7 wherein the higher is the pocked red blood cell count, the worse is the spleen function of the subject.

9. The method of claim 8 that comprises the steps of i) detecting the pocked red blood cells in the sample obtained from the patient, ii) comparing the count determined at step i)with a predetermined reference value, iii) and concluding that the patient has a good spleen function when the count quantified at step i) is lower than the predetermined reference value or concluding that the patient has a poor spleen function when the count determined at step i) is higher than the predetermined reference value.

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