WO2021224401A1

WO2021224401A1 - Methods and compositions for determining a reference range of β-galactose exposure platelet

Info

Publication number: WO2021224401A1
Application number: PCT/EP2021/062020
Authority: WO
Inventors: Alexandre KAUSKOT; Delphine Borgel; Dominique LASNE
Original assignee: INSERM (Institut National de la Santé et de la Recherche Médicale); Assistance Publique-Hôpitaux De Paris (Aphp); Université Paris-Saclay
Priority date: 2020-05-07
Filing date: 2021-05-06
Publication date: 2021-11-11

Abstract

The present invention relates to a method for determining a reference range of β-galactose exposure platelet in a biological sample obtained from a subject comprising the steps of: i) determining the concentration of Ricinus communis agglutin (RCA) and the value of Forward SCatter (FSC) in said biological sample; and ii) calculating the ratio between RCA and FSC. Inventors determined an optimal RCA concentration of 12.5 µg/mL for the assay. Importantly, the measure was stable for up to 4 hours (mean fluorescence intensity (MFI)-RCA: 1233±329 at T0h and 1480±410 at T4h). The platelet count did not induce a variation of RCA and the measure of RCA was stable when tested up to 24h after blood collection, demonstrating the robustness of the assay (MFI-RCA: 17 1252±434 at day 0 and 1140±297 24 hours after blood sampling). In order to take into account the platelet size, results should be expressed as RCA/FSC ratio. They then used the assay to study variability in platelet β-galactose exposure in a population of 120 healthy adults, they found that the ratio is independent of sex and blood group and they were able to define normal range, an important step before potential widespread use of the assay.

Description

METHODS AND COMPOSITIONS FOR DETERMINING A REFERENCE

RANGE OF B-GALACTOSE EXPOSURE PLATELET

FIELD OF THE INVENTION:

The invention is in the field of haematology. More particularly, the invention relates to methods and compositions for determining the quality of platelets.

BACKGROUND OF THE INVENTION:

Thrombocytopenia can have various causes, which are usually classified as being 3 central or peripheral. Techniques for exploring central thrombocytopenia (i.e. abnormal platelet production) include bone marrow assessment, plasma thrombopoietin assays, evaluation of the immature platelet fraction [1] As for peripheral causes of excessive platelet consumption they can be easily monitored. However, evidencing an increase in platelet clearance is more challenging because a routine laboratory assay for platelet clearance is not available. Indeed, the in vivo platelet half-life can only be measured in specialized centers after indiumlll labelling, and peripheral destruction can be revealed by a weak platelet transfusion yield. With a view to improving the diagnosis and treatment of thrombocytopenia, the search for new biomarkers prompted the assessment of platelet sialylation and b-galactose exposure, which were found to be regulators of platelet clearance. However, clinical laboratories do not have specific assays of platelet turnover related to b-galactose exposure. Sialic acids are terminal sugar components of glycoproteins and glycolipids and are incorporated during the sialylation process. In contrast, desialylation occurs during platelet ageing, with the cleavage of terminal sialic acids from platelet glycoproteins [2, 3]

The absence of sialic acid exposes b-galactose residues, which are considered to be senescence antigens. Their exposure influence platelet clearance via the cooperation between Ashwell-Morel receptors on hepatocytes and CLEC4F/MGL on Kupffer cells [4-6] Platelet b- galactose exposure can result from platelet hyposialylation or platelet desialylation. Both mechanisms have been described in various diseases or patients. For instance, mutations in the genes coding for the solute carrier family 35 member A1 (SLC35A1) sialic acid transporter and the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) have been linked to platelet hyposialylation and thrombocytopenia [7-13] Alternatively, platelet desialylation (via the activation of sialidases) associated with thrombocytopenia has been observed in cases of sepsis [14] and immune thrombocytopenia (ITP) [15-19] and following allogenic hematopoietic stem cell transplantation [20] Patients with ITP who present with significant platelet desialylation and anti-GPIb antibodies are less likely to respond to conventional first- line treatments [16] The sialidase inhibitor oseltamivir phosphate (used as an anti- influenza drug) reportedly increased the platelet count in a pediatric ITP patient who experienced platelet desialylation during an influenza virus A infection [19] and in an adult with chronic, anti-GPIb/IX-positive ITP [15] Furthermore, the combination of a sialidase inhibitor with drugs that boost platelet production induced a sustained platelet response in a proportion of patients with anti-GPIba-positive ITP and who had failed to respond to previous treatments [21]

The substantial elevation of the platelet count in this setting prompted interest in developping novel approached to the treatment of desialylation-related thrombocytopenia. However, prerequisites for the identification of thrombocytopenic diseases linked to desialylation/ -galactose exposure include the standardization of the b-galactose platelet assay and the determination of a reference range for b-galactose exposure in the healthy population. This reference range has not previously been determined. In mice, reference ranges are determined in animals with the same genetic background, and a comparison of b-galactose exposure on platelets exposed to various conditions is often sufficiently informative. However, the evaluation of a reference range for platelet desialylation in humans is much more challenging; a lack of knowledge of the variability within a healthy population precludes comparisons of patients with a single control subject. Moreover, the determination of reference values in a healthy population could help to identify patients with thrombocytopenia who might respond to sialidase inhibitors [15-19, 21] Accurate diagnosis of desialylation-related thrombocytopenia with a routine lab assay and the use of sialidase inhibitors might avoid the use of burdensome treatments, such as corticosteroids and splenectomy.

Correct diagnosis of the cause of thrombocytopenia is crucial for the appropriate management of patients. Hyposialylation/desialylation (characterized by abnormally high b- galactose exposure) accelerates platelet clearance and can lead to thrombocytopenia. However, the reference range for b-galactose exposure in healthy individuals has not previously been defined. Determination of this range is a prerequisite to the identification of thrombocytopenic diseases linked to changes in sialylation.

Accordingly, there is a need to find new tools to identify thrombocytopenic diseases linked to changes in sialylation.

SUMMARY OF THE INVENTION:

The invention relates to a method for determining a reference range of b-galactose exposure platelet in a biological sample obtained from a subject comprising the steps of: i) determining the concentration of Ricinus communis agglutin (RCA) and the value of Forward SCatter (FSC) in said biological sample; and ii) calculating the ratio between RCA and FSC.

In particular, the invention is defined by claims.

DETAILED DESCRIPTION OF THE INVENTION:

Inventors have developped a standardized flow cytometry assay of b-galactose exposure in platelet-rich plasma by using Ricinus communis agglutinin (RCA) for implementation in a clinical laboratory b-galactose exposure was measured in platelet-rich plasma by using flow cytometry and Ricinus communis agglutinin (RCA). A population of 120 healthy adults was recruited to study variability. They determined an optimal RCA concentration of 12.5 pg/mL. The measure was stable for up to 4 hours (mean fluorescence intensity (MFI)-RCA: 1233±329 at TOh and 1480±410 at T4h). The platelet count did not induce a variation of RCA and the measure of RCA was stable when tested up to 24h after blood collection (MFI-RCA: 1252±434 at day 0 and 1140±29724 hours after blood sampling). In order to take into account the platelet size, results should be expressed as RCA/FSC ratio. We used the assay to study variability in 120 healthy adults, and we found that the ratio is independent of sex and blood group.

Accordingly, they defined a normal range in a healthy population and several pre- analytical and analytical variables were evaluated, together with positive and negative controls. This assay may assist in the diagnosis of thrombocytopenic diseases linked to changes in b- galactose exposure.

Method for determining a reference range of b-salactose exposure

In a first aspect, the invention relates to a method for determining a reference range of b-galactose exposure platelet in a biological sample obtained from a subject comprising the steps of: i) determining the concentration of Ricinus communis agglutin (RCA) and the value of Forward SCatter (FSC) in said biological sample; and ii) calculating the ratio between RCA and FSC.

In a further embodiment, the method according to the invention further comprises a step of identifying non-specific populations appearing within the platelet population with a Mitotracker.

Typically, the invention relates to a method for determining a reference range of b- galactose exposure platelet in a biological sample obtained from a subject comprising the steps of: i) identifying non-specific populations appearing within the platelet population; ii) determining the concentration of Ricinus communis agglutin (RCA) and the value of Forward SCatter (FSC) in said biological sample; and ii) calculating the ratio between RCA and FSC.

As used herein, the term “subject” refers to any mammals, such as a rodent, a feline, a canine, and a primate. Particularly, in the present invention, the subject is a human. In a particular embodiment, the subject is a healthy subject.

As used herein, the term “platelet” also called as thrombocytes refers to a component of blood whose function is to react to bleeding from blood vessel injury by clumping, thereby initiating a blood clot. Platelets are made in bone marrow along with white and red blood cells. A normal platelet count is 150,000 to 450,000 platelets per microliter of blood.

As used herein, the term “b-galactose” also known as galactose, beta-D-galactose or beta-D-galactopyranose refers to a monosaccharide sugar that is about as sweet as glucose. Galactose molecule linked with a glucose molecule forms a lactose molecule b-galactose is released by an enzyme reaction: B-galactosidase hydrolyzes b-D-galactoside bonds to release D-galactose, and is generally present in microorganisms, animals and plants. Typically, b- galactose has the following chemical formula in the art CeHuOe and CAS number: 7296-64-2.

As used herein, the term “platelet b-galactose exposure” refers to platelet which have b- galactose on their surface.

As used herein, the term biological sample refers to any sample obtained from a subject, such as a serum sample, a plasma sample, a urine sample, a blood sample, a lymph sample, or a biopsy. In the context of the invention, the biological sample is a blood sample. In a particular embodiment, the biological sample is plasma sample. More particularly, the biological sample is venous blood sample. Typically, the venous blood is collected in vacutainer® containing 7.5% K3 EDTA. Platelet-rich plasma (PRP) is obtained after two centrifugation steps, i.e. 10 minutes at 38 g and then 10 minutes at 122 g, to enable the recovery of large and giant platelets.

As used herein, the term “non-specific populations appearing within the platelet population” refers to impurities and debris present in the biological sample. Said impurities refer to non-specific population. Typically, this step allows to eliminate elements in the blood that may or may not have mitochondria. For example, red blood cells do not have mitochondria. This will be useful to know the background noise before calculation the ratio RCA/FSC.

Typically, this step is performed by MitoTracker™ Deep Red FM. MitoTracker^® Red FM is a far red-fluorescent dye (abs/em -581/644 nm) that stains mitochondria in live cells and its accumulation is dependent upon membrane potential. As used herein, the term “Ricinus communis agglutin” (RCA) refers to a lectin which binds to b-galactose. This lectin consists of two subunits of 60 kDa which can be dissociated by reducing agents into closely related chains between 27 kDa and 33 kDa. One of the chains appears to be common to the “B” chain of another castor bean lectin, ricin, while the other chain is unique to RCA I. The B chain binds to galactose or N-acetylgalactosamine residues of membrane glycoconjugates. RCA is measured by flow cytometry. In the context of the invention, said RCA is also called as RCAI or RCA120. Typically, platelet surface b-galactose exposure was determined using FITC-conjugated RCA (Vector Labs, Burlingame, CA). Briefly, PRP diluted was incubated with RCA for 30 min at room temperature. The reaction was stopped with PBS and then analyzed using flow cytometry. In another embodiment, the samples in which RCA had been incubated with b-lactose were used as negative control. As a positive control of platelet desialylation (i.e. the maximum possible amount of b-galactose exposure due to sialic acid removal from the platelet surface), platelet rich plasma (PRP) was incubated with 0.5 U/mL neuraminidase from Clostridium perfringens (NeuC, Sigma- Aldrich). The stability of platelet b-galactose exposure was studied from 10 controls. On the day of sampling, each blood sample was divided into two aliquots; one was assayed in the two hours, and the other was assayed after 24 hours of storage at room temperature. The stability of RCA binding was evaluated 30 minutes and 1, 2, 3 and 4 hours after the start of the incubation. In each sample, the geometric mean fluorescence intensity (MFI) was determined for a total of 5,000 platelets.

In a particular embodiment, the concentration of RCA is 12.5 pg/mL.

In a further embodiment, the method according to the invention, wherein the concentration or quality of RCA can be controlled in lyophilized platelets that are desialylated. Typically, platelets are resuspended at a concentration of 150 G/L in PBS and then diluted at 50G/L in the assay. Platelets are then stained with the RCA-FITC at 12.5 pg/mL. Two vials of RCA-FITC from the same lot were then used with a delay of opening of 12 months. Each vial is referred as old (opened 12 months ago) and young vial (recently opened). The signal of RCA- FITC decreased by 2.5 fold times after 12 months of storage. Accordingly, the fixed lyophilized platelets can be used as quality control of RCA. A decrease of ±< 10% of the MFI of the RCA- FITC of same RCA vial has to be considered to rule out the vial.

As used herein, the term “Forward Scatter” (FSC) refers to the amount of laser light that passes around the cell, and is proportional to the cell’s diameter. FSC is measured during flow cytometry experiment. Flow cytometry is a method of single-cell analysis that includes the characterization of a cell's physical properties.

Typically, on the day when the blood sample was collected, the FSC (the amount of laser light that passes around the cell, and is proportional to the cell’s diameter) is measured and the levels of two conventional glycoproteins (GPIba and GPIIb) proportional with the mean platelet volume, at the same time as RCA binding. The RCA/size ratios (RCA/FSC, RCA/GPIba and RCA/GPIIb) are stable over the 4 hours of measurement.

As used herein, the term “value” refers to the number of cells passed through out of the laser.

As used herein, the term “ratio” refers to the relationship between two amounts. Typically, in the context of the invention, ratio refers to the relationship between RCA and FSC as defined above. Typically, the ratio RCA/FSC ratio is determined in a population of healthy subjects. More particularly, the ratio is independent of sex and blood group. In another embodiment, the ratio comprises between 0.237 to 0.941.

In a particular embodiment, the ratio of RC/FSC is also called as reference range when it is determined in a population of healthy subjects.

In the context of the invention, the ratio as determined above is the reference range.

Typically, the ratio or reference range according to the invention comprising between 0.237 to 0.941.

In a further embodiment, the ratio or the reference range calculated in a healthy population according to the invention is: 0.237, 0.238, 0.239, 0.240, 0.241, 0.242, 0.243, 0.244, 0.245, 0.246, 0.247, 0.248, 0.249, 0.250, 0.251, 0.252, 0.253, 0.254, 0.255, 0.256, 0.257, 0.258,

0.259, 0.260, 0.261, 0.262, 0.263, 0.264, 0.265, 0.266, 0.267, 0.268, 0.269, 0.270, 0.271, 0.272,

0.273, 0.274, 0.275, 0.276, 0.277, 0.278, 0.279, 0.280, 0.281, 0.282, 0.283, 0.284, 0.285, 0.286,

0.287, 0.288, 0.289, 0.290 , 0.291, 0.292, 0.293, 0.294, 0.295, 0.296, 0.297, 0.298, 0.299, 0.300, 0.301, 0.302, 0.303, 0.304, 0.305, 0.306, 0.307, 0.308, 0.309, 0.310, 0.311, 0.312, 0.313, 0.314,

0.315, 0.316, 0.317, 0.318, 0.319, 0.320, 0.321, 0.322, 0.323, 0.324, 0.325, 0.326, 0.327, 0.328,

0.329, 0.330, 0.331, 0.332, 0.333, 0.334, 0.335, 0.336, 0.337, 0.338, 0.339, 0.340, 0.341, 0.342,

0.343, 0.344, 0.345, 0.346, 0.347, 0.348, 0.349, 0.350, 0.351, 0.352, 0.353, 0.354, 0.355, 0.356,

0.357, 0.358, 0.359, 0.360, 0.361, 0.362, 0.363, 0.364, 0.365, 0.366, 0.367, 0.368, 0.369, 0.370,

0.371, 0.372, 0.373, 0.374, 0.375, 0.376, 0.377, 0.378, 0.379, 0.380, 0.381, 0.382, 0.383, 0.384,

0.385, 0.386, 0.387, 0.388, 0.389, 0.390, 0.391, 0.392, 0.393, 0.394, 0.395, 0.396, 0.397, 0.398,

0.399, 0.400, 0.401, 0.402, 0.403, 0.404, 0.405, 0.406, 0.407, 0.408, 0.409, 0.410, 0.411, 0.412,

0.413, 0.414, 0.415, 0.416, 0.417, 0.418, 0.419, 0.420, 0.421, 0.422, 0.423, 0.424, 0.425, 0.426, 0.427, 0.428, 0.429, 0.430, 0.431, 0.432, 0.433, 0.434, 0.435, 0.436, 0.437, 0.438, 0.439, 0.440,

0.441, 0.442, 0.443, 0.444, 0.445, 0.446, 0.447, 0.448, 0.449, 0.450, 0.451, 0.452, 0.453, 0.454,

0.455, 0.456, 0.457, 0.458, 0.459, 0.460, 0.461, 0.462, 0.463, 0.464, 0.465, 0.466, 0.467, 0.468,

0.469, 0.470, 0.471, 0.472, 0.473, 0.474, 0.475, 0.476, 0.477, 0.478, 0.479, 0.480, 0.481, 0.482,

0.483, 0.484, 0.485, 0.486, 0.487, 0.488, 0.489, 0.490, 0.491, 0.492, 0.493, 0.494, 0.495, 0.496,

0.497, 0.498, 0.499, 0.500, 0.501, 0.502, 0.503, 0.504, 0.505, 0.506, 0.507, 0.508, 0.509, 0.510,

0.511, 0.512, 0.513, 0.514, 0.515, 0.516, 0.517, 0.518, 0.519, 0.520, 0.521, 0.522, 0.523, 0.524,

0.525, 0.526, 0.527, 0.528, 0.529, 0.530, 0.531, 0.532, 0.533, 0.534, 0.535, 0.536, 0.537, 0.538,

0.539, 0.540, 0.541, 0.542, 0.543, 0.544, 0.545, 0.546, 0.547, 0.548, 0.549, 0.550, 0.551, 0.552,

0.553, 0.554, 0.555, 0.556, 0.557, 0.558, 0.559, 0.560, 0.561, 0.562, 0.563, 0.564, 0.565, 0.566,

0.567, 0.568, 0.569, 0.570, 0.571, 0.572, 0.573_; 0.574, 0.575, 0.576, 0.577, 0.578, 0.579, 0.580

0.581, 0.582, 0.583, 0.584, 0.585, 0.586, 0.587, 0.588, 0.589, 0.590, 0.591, 0.592, 0.593, 0.594,

0.595, 0.596, 0.597, 0.598, 0.599, 0.600, 0.601, 0.602, 0.603, 0.604, 0.605, 0.606, 0.607, 0.608,

0.609, 0.610, 0.611, 0.612, 0.613, 0.614, 0.615, 0.616, 0.617, 0.618, 0.619, 0.620, 0.621, 0.622,

0.623, 0.624, 0.625, 0.626, 0.627, 0.628, 0.629, 0.630, 0.631, 0.632, 0.633, 0.634, 0.635, 0.636,

0.637, 0.638, 0.639, 0.640, 0.641, 0.642, 0.643, 0.644, 0.645, 0.646, 0.647, 0.648, 0.649, 0.650,

0.651, 0.652, 0.653, 0.654, 0.655, 0.656, 0.657, 0.658, 0.659, 0.660, 0.661, 0.662, 0.663, 0.664,

0.665, 0.666, 0.667, 0.668, 0.669, 0.670, 0.671, 0.672, 0.673, 0.674, 0.675, 0.676, 0.677, 0.678,

0.679, 0.680, 0.681, 0.682, 0.683, 0.684, 0.685, 0.686, 0.687, 0.688, 0.689, 0.690, 0.691, 0.692,

0.693, 0.694, 0.695, 0.696, 0.697, 0.698, 0.699, 0.700, 0.701, 0.702, 0.703, 0.704, 0.705, 0.706,

0.707, 0.708, 0.709, 0.710, 0.711, 0.712, 0.713, 0.714, 0.715, 0.716, 0.717, 0.718, 0.719, 0.720,

0.721, 0.722, 0.723, 0.724, 0.725, 0.726, 0.727, 0.728, 0.729, 0.730, 0.731, 0.732, 0.733, 0.734,

0.735, 0.736, 0.737, 0.738, 0.739, 0.740, 0.741, 0.742, 0.743, 0.744, 0.745, 0.746, 0.747, 0.748,

0.749, 0.750, 0.751, 0.752, 0.753, 0.754, 0.755, 0.756, 0.757, 0.758, 0.759, 0.760, 0.761, 0.762,

0.763, 0.764, 0.765, 0.766, 0.767, 0.768, 0.769, 0.770, 0.771, 0.772, 0.773, 0.774, 0.775, 0.776,

0.777, 0.778, 0.779, 0.780, 0.781, 0.782, 0.783, 0.784, 0.785, 0.786, 0.787, 0.788, 0.789, 0.790,

0.791, 0.792, 0.793, 0.794, 0.795, 0.796, 0.797, 0.798, 0.799, 0.800, 0.801, 0.802, 0.803, 0.804,

0.805, 0.806, 0.807, 0.808, 0.809, 0.810, 0.811, 0.812, 0.813, 0.814, 0.815, 0.816, 0.817, 0.818,

0.819, 0.820, 0.821, 0.822, 0.823, 0.824, 0.825, 0.826, 0.827, 0.828, 0.829, 0.830, 0.831, 0.832,

0.833, 0.834, 0.835, 0.836, 0.837, 0.838, 0.839, 0.840, 0.841, 0.842, 0.843, 0.844, 0.845, 0.846,

0.847, 0.848, 0.849, 0.850, 0.851, 0.852, 0.853, 0.854, 0.855, 0.856, 0.857, 0.858, 0.859, 0.860,

0.861, 0.862, 0.863, 0.864, 0.865, 0.866, 0.867, 0.868, 0.869, 0.870, 0.871, 0.872, 0.873, 0.874,

0.875, 0.876, 0.877, 0.878, 0.879, 0.880, 0.881, 0.882, 0.883, 0.884, 0.885, 0.886, 0.887, 0.888,

0.889, 0.890, 0.891, 0.892, 0.893, 0.894, 0.895, 0.896, 0.897, 0.898, 0.899, 0.900, 0.901, 0.902, 0.903, 0.904, 0.905, 0.906, 0.907, 0.908, 0.909, 0.910, 0.911, 0.912, 0.913, 0.914, 0.915, 0.916, 0.917, 0.918, 0.919, 0.920, 0.921, 0.922, 0.923, 0.924, 0.925, 0.926, 0.927, 0.928, 0.929, 0.930, 0.931, 0.932, 0.933, 0.934, 0.935, 0.936, 0.937, 0.938, 0.939, 0.940 or 0.941.

In a further embodiment, the method according to the invention is suitable to measure the number of platelet b-galactose exposure in a biological sample.

Accordingly, the invention relates to method according to the invention is suitable to measure the number of platelet b-galactose exposure in a biological sample obtained from a subject comprising the steps of: i) identifying non-specific populations appearing within the platelet population; ii) determining the concentration of Ricinus communis agglutin (RCA) and the value of Forward SCatter (FSC) in said biological sample; and ii) calculating the ratio between RCA and FSC.

Uses of the method according to the invention

The method as described above is suitable to measure the quality of platelets.

Accordingly, in a second aspect, the invention relates to a method for determining the quality of platelets in a biological sample comprising the steps of i) calculating the ratio between RCS and FSC as described above; and ii) comparing said ratio with a reference range.

Typically, the invention relates to a method for determining the quality of platelets in a biological sample comprising the steps of: i) identifying non-specific populations appearing within the platelet population; ii) determining the concentration of RCA and the value of FSC in said biological sample; iii) calculating the ratio between RCA and FSC; iv) comparing said ratio with a reference range; and, v) concluding that the quality of platelets is stable and/or maintained when the the ratio calculated is in the reference range or concluding that the quality of platelets is not stable and/or maintained when the ratio is outside the reference range.

In a particular embodiment, the method according to the invention, wherein the concentration or quality of RCA is controlled in lyophilized platelets as described above.

In a further embodiment, the method according to the invention is suitable to measure the quality of platelets during a transfusion. As used herein, the term “quality” refers to maintain of platelets’ functions in a biological sample. Typically, it is known in the art that human platelets have a half-life of 8 to 9 days and 150,000 to 450,000 platelets per microliter of blood in the circulation. Platelets’ function is to react to bleeding from blood vessel injury by clumping, thereby initiating a blood clot. The quality of platelets is determined by measuring the ratio between RCA and FSC according to the invention and compared said ratio to reference range as described above.

When the ratio calculated is in the reference range, it means that the quality of the platelets is stable and/or maintained. More particularly, the platelets’ functions are conserved.

When the ratio is outside the reference range (inferior to 0.237 or superior to 0.941), it means that the quality of the platelets is not stable and/or maintained. More particularly, the platelets’ functions are not conserved.

As used herein, the term biological sample refers to any sample obtained from a subject, such as a serum sample, a plasma sample, a urine sample, a blood sample, a lymph sample, or a biopsy. In the context of the invention, the biological sample is a blood sample. More particularly, the biological sample is venous blood sample. In another embodiment, the biological sample is a venous blood sample obtained from a donor. Typically, the biological sample is a storage bag for a transfusion.

Typically, the quality of platelets can also be measured before a transfusion to a recipient.

Platelet transfusion is most frequently used to correct unusually low platelet counts, either to prevent spontaneous bleeding (typically at counts below 10xl0⁹/L) or in anticipation of medical procedures that will necessarily involve some bleeding.

Platelets are either isolated from collected units of whole blood and pooled to make a therapeutic dose, or collected by platelet apheresis: blood is taken from the donor, passed through a device which removes the platelets, and the remainder is returned to the donor in a closed loop.

As used herein, the term “quality” refers to maintain of platelets’ functions during the storage period for a transfusion.

As used herein, the term “transfusion” refers to an event where blood is removed from one individual (donor), animal, or human, and transfused to another in need (recipient).

As used herein, the term “donor” refers to a human or animal, donating blood.

In a particular embodiment, the donor is a healthy human.

In another embodiment, the donor is a healthy animal. As used herein, the term “recipient” refers to a corresponding human or animal receiving blood.

In a further embodiment, the recipient is a subject suffering from thrombocytopenia (elicited by chemotherapy and/or other diseases).

In another embodiment, the recipient is an elderly person.

In another embodiment, the recipient is pregnant.

In another embodiment, the recipient is a human susceptible to have a surgery.

In another embodiment, the recipient is a human susceptible to have an organ transplantation.

In a third aspect, the invention relates to a method for determining whether platelets in a biological sample are desialylated or hyposialylated comprising the steps of: i) calculating the ratio between RCA and FSC as described above; and ii) concluding that platelets are desialylated or hyposialylated when the ratio is outside the reference range according to the invention.

Typically, the invention relates to the invention relates to a method for determining whether platelets in a biological sample obtained from a subject are desialylated or hyposialylated comprising the steps of: i) identifying non-specific populations appearing within the platelet population; ii) determining the concentration of RCA and the value of FSC in said biological sample; ii) calculating the ratio between RCA and FSC; iii) comparing said ratio with a reference range; and iv) concluding that platelets are desialylated or hyposialylated when the ratio is outside the reference range according to the invention.

As used herein, the terms “desialylated”, “hyposialyted” desialylation or “hyposialation” refer to any reaction that deplete a sialyl acid from a molecule. Sialic acids are terminal sugar components of glycoproteins and glycolipids and are incorporated during the sialylation process. Sialic acids are monosaccharides with a shared 9-carbon backbone, and they are typically found at the outermost end of the glycan chains in all cell types. Sialic acids play pivotal roles in many physiologically and pathologically important processes. They play numerous roles in several aspects of immunity, and they might serve as “self’ markers in different physiological and pathophysiological functions. Typically, the hyposialylation/desialylation on platelets is characterized by abnormally high b-galactose exposure that accelerates platelet clearance and can lead to many diseases such as thrombocytopenia.

As used herein, the term “outside the reference range” refers to when the ratio is inferior to 0.237 or superior to 0.941.

Typically, when the ratio calculated is in the reference range, it means that that platelets are not desialylated or hyposialylated.

Typically, when the ratio is outside the reference range (inferior to 0.237 or superior to 0.941), it means that that platelets are desialylated or hyposialylated.

In another embodiment, the invention a method for determining whether platelets in a biological sample are hypersialylated comprising the steps of: i) calculating the ratio between RCA and FSC as described above; and ii) concluding that platelets are hypersialylated when the ratio is inferior to the reference range according to the invention.

Typically, the invention relates to the invention relates to a method for determining whether platelets in a biological sample obtained from a subject are hypersialylated comprising the steps of: i) identifying non-specific populations appearing within the platelet population; ii) determining the concentration of RCA and the value of FSC in said biological sample; ii) calculating the ratio between RCA and FSC; iii) comparing said ratio with a reference range; and iv) concluding that platelets are hypersialylated when the ratio is inferior to the reference range according to the invention.

As used herein, the term “hypersialylation” refers to an increase of the immature platelet fraction or young platelets.

Typically, when the ratio calculated is inferior to the reference range (e.g. inferior to 0.237), it means that subject the platelets are hypersialylated.

The method according to the invention is suitable to diagnosis a platelet desialylation related disease.

Accordingly, in a forth aspect, the invention relates to a method for diagnosing a platelet desialylation related disease in a subject comprising the steps of: i) calculating the ratio between RCA and FSC as described above; and ii) concluding that the subject is suffering or is susceptible to suffer from a platelet desialylation related disease when the ratio is outside the reference range according to the invention. Typically, the invention relates to the invention relates to a method for diagnosing a platelet desialylation related disease in a subject comprising the steps of: i) identifying non-specific populations appearing within the platelet population; ii) determining the concentration of RCA and the value of FSC in said biological sample; iii) calculating the ratio between RCA and FSC; iv) comparing said ratio with a reference range; and v) concluding that the subject is suffering or is susceptible to suffer from a platelet desialylation related disease when the ratio is outside the reference range according to the invention.

As used herein term "diagnosing" refers to classifying a disease or a symptom, determining a severity of the disease, monitoring disease progression, forecasting an outcome of a disease and/or prospects of recovery.

As used herein, the term biological sample refers to any sample obtained from a subject, such as a serum sample, a plasma sample, a urine sample, a blood sample, a lymph sample, or a biopsy. In the context of the invention, the biological sample is a blood sample.

In a particular embodiment, the biological sample is plasma sample.

In another embodiment, the biological sample is plasma sample with platelets called Platelet-Rich Plasma (PRP). More particularly, the biological sample is venous blood sample.

In the context of the invention, the biological sample is obtained from thrombocytopenic suspected samples.

In a particular embodiment, the non-specific populations appearing within the platelet population is determined by Mitotracker. This will be useful to know the background noise before calculation the ratio RCA/FSC in thrombocytopenic suspected subjects.

As used herein term “platelet desialylation related disease” refers to diseases elicited by platelet desialylation.

Typically, when the ratio calculated is in the reference range (between 0.237 and 0.941), it means that subject does not suffer from a platelet desialylation related disease.

Typically, when the ratio is outside the reference range (inferior to 0.237 or superior to 0.941), it means that the subject suffers from a platelet desialylation related disease.

In a particular embodiment, the platelet desialylation related disease is selected from the group consisting of but not limited to: thrombocytopenia, sepsis, immune thrombocytopenia (ITP), gestational thrombocytopenia, congenital disorders of glycosylation, VWF-type 2B disease, bacterial infections, viral infection (e.g. influenza virus A infection, dengue), parasitic infection (e.g. malaria), liver diseases (e.g. NAFLD, NASH), allogenic hematopoietic stem cell transplantation, disorders related to Trans Aortic Valve Implantation (TAVI) or Mechanical Circulatory Support Implantation (MCSI).

In a particular embodiment, the platelet desialylation related disease is thrombocytopenia.

Thrombocytopenia is characterized by abnormally low levels of platelets in the blood. Thrombocytopenia might occur as a result of a bone marrow disorder such as leukemia or an immune system problem. Or it can be a side effect of taking certain medications. Thrombocytopenia signs and symptoms may include: easy or excessive bruising (purpura), superficial bleeding into the skin that appears as a rash of pinpoint-sized reddish-purple spots (petechiae), usually on the lower legs, prolonged bleeding from cuts, bleeding from gums or nose, blood in urine or stools, unusually heavy menstrual flows, fatigue or enlarged spleen. Factors that can decrease platelet production include: leukemia and other cancers, some types of anemia, viral infections, such as hepatitis C or HIV, chemotherapy drugs and radiation therapy or heavy alcohol consumption.

In a particular embodiment, the thrombocytopenia is elicited by chemotherapy agents used for treating a cancer.

As used herein, the term "chemotherapeutic agent" refers to chemical compounds that are effective in inhibiting tumor growth. Examples of chemotherapeutic agents include multkinase inhibitors such as sorafenib and sunitinib, alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolo melamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CBI-TMI); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimus tine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as the enediyne antibiotics (e.g. calicheamicin, especially calicheamicin (11 and calicheamicin 211, see, e.g., Agnew Chem Inti. Ed. Engl. 33: 183-186 (1994); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, canninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6- diazo-5-oxo-L-norleucine, doxorubicin (including morpholino- doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idanrbicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptomgrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti- adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defo famine; demecolcine; diaziquone; elfomithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; pento statin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; rhizoxin; sizofiran; spirogennanium; tenuazonic acid; triaziquone; 2, 2', 2"- trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridinA and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobromtol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.].) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6- thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisp latin and carhop latin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-1 1 ; topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are antihormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

In a particular embodiment, the platelet desialylation related disease is idiopathic or Immune thrombocytopenia (ITP). It is also known as immune (idiopathic) thrombocytopenic purpura (ITP), refers to a clinical disorder in which thrombocytopenia manifests as a bleeding tendency, purpura, or petechiae.

Desialylation-related thrombocytopenia might also be present in other acquired disorders related to VWF-platelet binding during common clinical procedures, such as the implantation of transaortic valves and mechanical circulatory support.

Accordingly, in another embodiment, the platelet desialylation related disease is a disorder related to Trans Aortic Valve Implantation (TAVI). As used, herein, the term “TAVI” also referred to as transcatheter aortic valve replacement (TAVR), is a procedure that replaces diseased aortic valve with a man-made valve. The aortic valve usually opens when blood is pumped from heart to the rest of body. Aortic stenosis is a condition where the aortic valve cannot open and close properly. This condition puts extra strain on heart and can result in breathlessness, swollen ankles, chest pain, dizziness, and sometimes, blackouts.

In another embodiment, the platelet desialylation related disease is a disorder related to Mechanical Circulatory Support Implantation (MCSI). MSCI refers to devices that assist the ventricle and are attached to it in use. These are called Ventricular Assist Devices (VADs), and are designed to drive a flow of blood that is in parallel with flow within the native heart, between the ventricle and the aorta. In other words, they are designed as left (or right) ventricular assist devices (LVADs or RVADs), pumping devices that directly unload the respective ventricle.

In another embodiment, the platelet desialylation related disease is allogeneic hematopoietic cell transplantation (HSCT) related disease. HSCT refers to the transplantation of hematopoietic stem cells, usually derived from bone marrow, peripheral blood, or umbilical cord blood. It may be autologous (the patient's own stem cells are used), allogeneic (the stem cells come from a donor) or syngeneic (from an identical twin). In the context of the invention, the hematopoietic stem cells are from a donor (allogenic). Progress in allogeneic HSCT depends on several factors, including the adequate prevention and management of associated complications, advances in the conventional management of diseases currently treated with allogeneic HSCT, expansion of the donor pool, selective control of GVHD, development of more effective preparative regimens to eradicate the neoplastic cell population, characterization of anew generation of hematopoietic growth factors and cytokines, and development of newer techniques for ex vivo manipulation of stem cells.

In another embodiment, the platelet desialylation related disease is liver diseases.

As used herein, the term “liver disease” refers to any disturbance of liver function that causes illness. The liver is responsible for many critical functions from protein production and blood clotting to cholesterol, glucose (sugar), and iron metabolism. When it becomes diseased or injured, the loss of those functions can cause significant damage to the body. Liver disease is also referred to as hepatic disease. The liver disease is selected from the group consisting of: Nonalcoholic fatty liver disease (NAFLD), Non-alcoholic steatohepatitis (NASH), or fibrotic NASH.

In another embodiment, the platelet desialylation related disease is VWF-type 2B.

As used herein, the term “VWF-type 2B” refers to a subtype of type 2 VWD characterized by a bleeding disorder associated with an increase in the affinity of the Willebrand factor (von Willebrand factor; VWF) for platelets. This anomaly results in spontaneous binding of high molecular weight VWF multimers to platelets leading to rapid clearance of both the platelets (increasing the risk of thrombocytopenia) and the high molecular weight VWF multimers from the plasma.

In another embodiment, the platelet desialylation related disease is sepsis.

As used herein, the term “sepsis” has its general meaning in the art and is used to identify the continuum of the clinical response to infection. Patients with sepsis present evidences of infection and clinical manifestations of inflammation. Sepsis is defined as SIRS secondary to documented or suspected infection.

In another embodiment, the platelet desialylation related disease is viral infection.

In a further embodiment, the platelet desialylation related disease is an influenza virus A infection.

As used herein, the term "influenza virus type A infection" refers to any infection caused by an influenza virus type A without consideration of serotype based on hemagglutinine (HI to H 15) and neuraminidase (N1 to N9) expression. Examples of influenza virus type A include but are not limited to H1N1, H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3, and H10N7.

In a further embodiment, the platelet desialylation related disease is dengue virus infection. As used herein, the term “dengue virus” as is general meaning in the art and denotes an RNA virus of the family Flaviviridae; genus Flavivirus. Other members of the same genus include yellow fever virus, West Nile virus, St. Louis encephalitis virus, Japanese encephalitis virus, tick-home encephalitis virus, Kyasanur forest disease virus, and Omsk hemorrhagic fever virus. Most are transmitted by arthropods (mosquitoes or ticks), and are therefore also referred to as arboviruses (arthropod-borne viruses). According to the invention, the dengue virus may be of any serotype, i.e. serotype 1, 2, 3 or 4.

In a further embodiment, the platelet desialylation related disease is parasitic infection.

In a further embodiment, the platelet desialylation related disease is malaria.

As used herein, the term “malaria” refers to a parasitic disease caused by a protozoon of the genus Plasmodium. The five subspecies active in humans are P. falciparum, P. vivax, P. malariae, P. ovale, and P. knowlesiwithP. falciparumbeing responsible for most malaria-related death.

As used herein, the term "subject" refers to any mammals, such as a rodent, a feline, a canine, and a primate. Particularly, in the present invention, the subject is a human.

In a particular embodiment, the subject is a human who is susceptible to have a platelet desialylation related disease.

In a particular embodiment, the subject is a human who is susceptible to have thrombocytopenia.

In a particular embodiment, the subject is a human who is susceptible to have sepsis.

In a particular embodiment, the subject is a human who is susceptible to have immune thrombocytopenia (ITP).

In a particular embodiment, the subject is a human who is susceptible to have allogenic hematopoietic stem cell transplantation.

In a particular embodiment, the subject is a human who is susceptible to have a sepsis.

In a particular embodiment, the subject is a human who is susceptible to have an influenza virus A infection.

In a particular embodiment, the subject is a human who is susceptible to have a dengue virus infection.

In a particular embodiment, the subject is a human who is susceptible to have malaria.

In a particular embodiment, the subject is a human who is susceptible to have VWF type 2B.

In a particular embodiment, the subject is a human who is susceptible to have liver disease such as NAFLD or NASH. In a particular embodiment, the subject is a human who is susceptible to have disorder related to Trans Aortic Valve Implantation (TAVI) or Mechanical Circulatory Support Implantation (MCSI).

The method according to the invention is suitable to diagnosis a platelet hypersialylation related disease.

In a particular embodiment, the method for diagnosing a platelet hypersialylation related disease in a subject comprising the steps of: i) calculating the ratio between RCA and FSC as described above; and ii) concluding that the subject is suffering or is susceptible to suffer from a platelet hypersialylation related disease when the ratio is inferior to the reference range according to the invention.

Typically, the invention relates to the invention relates to a method for diagnosing a platelet hypersialylation related disease in a subject comprising the steps of: i) identifying non-specific populations appearing within the platelet population; ii) determining the concentration of RCA and the value of FSC in said biological sample; iii) calculating the ratio between RCA and FSC; iv) comparing said ratio with a reference range; and v) concluding that the subject is suffering or is susceptible to suffer from a platelet hypersialylation related disease when the ratio is inferior the reference range according to the invention.

Typically, the ratio is inferior to 0.237.

In the context of the invention, the platelet hypersialylation-related diseases could be observed in case of compensation mechanisms from the bone marrow to recover in thrombocytopenic patients a normal platelet count. This boost of the bone marrow stimulates the production of young platelets also called immature platelet fraction that are hypersialylated compared to old platelets.

In a fifth aspect, the invention relates to a method for predicting whether a subject suffering from a platelet desialylation related disease will achieve a response with a sialidase inhibitor comprising the steps: i) calculating the ratio between RCA and FSC as described above; and ii) concluding that the subject will achieve a response with a sialidase inhibitor when the ratio is in the reference range according to the invention or concluding that the subject will not achieve a response with a sialidase inhibitor when the ratio is outside the reference range according to the invention. Typically, the invention relates to the invention relates to a method for predicting whether a subject suffering from a platelet desialylation related disease will achieve a response with a sialidase inhibitor comprising the steps of: i) identifying non-specific populations appearing within the platelet population; ii) determining the concentration of RCA and the value of FSC in said biological sample; iii) calculating the ratio between RCA and FSC; iv) comparing said ratio with a reference range; and v) concluding that the subject will achieve a response (responder) with a sialidase inhibitor when the ratio is in the reference range according to the invention or concluding that the subject will not achieve a response with a sialidase inhibitor when the ratio is outside the reference range according to the invention.

As used herein, the term "predicting" means that the subject to be analyzed by the method of the invention is allocated either into the group of subjects who will achieve a response, or into a group of subjects who will not achieve as response for the treatment with a sialidase inhibitor.

As used herein the terms “will achieve a response” or “responder” in the context of the invention refer to a subject that will achieve a response, i.e. a subject where the platelet desialylation related disease is eradicated, reduced or improved. According to the invention, the responders have an objective response and therefore the term does not encompass subjects having a stabilized platelet desialylation related disease such that the disease is not progressing after the treatment with the sialidase inhibitor. A non- responder or refractory patient includes subjects for whom the platelet desialylation related disease does not show reduction or improvement after the treatment with the sialidase inhibitor. According to the invention the term“non-responder” also includes subjects having a stabilized platelet desialylation related disease. Typically, the characterization of the subject as a responder or non-responder can be performed by reference to a standard or a training set. The standard may be the profile of a subject who is known to be a responder or non-responder or alternatively may be a numerical value. Such predetermined standards may be provided in any suitable form, such as a printed list or diagram, computer software program, or other media.

Typically, when the ratio calculated is in the reference range (between 0.237 and 0.41), it means that subject will achieve a response with a sialidase inhibitor. Typically, when the ratio is outside the reference range (inferior to 0.237 or superior to 0.941), it means that the subject will not achieve a response with a sialidase inhibitor.

In a particular embodiment, the subject is a human who is susceptible to have liver disease such as NAFLD or NASH.

In a particular embodiment, the subject is a human who is susceptible to have disorder related to Trans Aortic Valve Implantation (TAVI) or Mechanical Circulatory Support Implantation (MCSI).

As used herein, the term “sialidase” also called as neuraminidase refers to enzymes which are glycoside hydrolase enzymes that cleave the glycosidic linkages of neuraminic acids. Neuraminidase enzymes are a large family, found in a range of organisms. Human sialidase homologues have been described in the human genome (NEU1, NEU2, NEU3, NEU4).

As used herein, the term “sialidase inhibitor” refers to a natural or synthetic compound that has a biological effect to inhibit or significantly reduce the activity or expression of the transcripts and/or proteins. Thus, a "sialidase inhibitor" refers to a natural or synthetic compound that has a biological effect to inhibit or significantly reduce the activity or expression of sialidase transcripts and/or proteins.

Typically, the inhibitor of sialidase expression has a biological effect on one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5' cap formation, and/or 3' end formation); (3) translation of an RNA into a polypeptide or protein; and/or (4) post-translational modification of a polypeptide or protein.

Typically, the inhibitor of sialidase activity has a biological effect on the binding or activity of sialidase. Inhibition can be partial or total, resulting in a reduction or modulation in the activity of the enzyme as detected.

In a particular embodiment, the sialidase inhibitor activity is a peptide, petptidomimetic, small organic molecule, antibody or aptamers. The term “peptidomimetic” refers to a small protein-like chain designed to mimic a peptide. In a particular embodiment, the inhibitor of sialidase is an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity.

In some embodiments, the sialidase inhibitor activity is an antibody. As used herein, the term “antibody” is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity. The term includes antibody fragments that comprise an antigen binding domain such as Fab', Fab, F(ab')2, single domain antibodies (DABs), TandAbs dimer, Fv, scFv (single chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies, diabodies, bispecific antibody fragments, bibody, tribody (scFv-Fab fusions, bispecific or trispecific, respectively); sc-diabody; kappa(lamda) bodies (scFv-CL fusions); BiTE (Bispecific T-cell Engager, scFv-scFv tandems to attract T cells); DVD-Ig (dual variable domain antibody, bispecific format); SIP (small immunoprotein, a kind of minibody); SMIP ("small modular immunopharmaceutical" scFv-Fc dimer; DART (ds-stabilized diabody "Dual Affinity ReTargeting"); small antibody mimetics comprising one or more CDRs and the like. The techniques for preparing and using various antibody-based constructs and fragments are well known in the art (see Rabat et ak, 1991, specifically incorporated herein by reference). Diabodies, in particular, are further described in EP 404, 097 and WO 93/1 1 161; whereas linear antibodies are further described in Zapata et al. (1995). Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques or can be chemically synthesized. Techniques for producing antibody fragments are well known and described in the art. For example, each of Beckman et ak, 2006; Holliger & Hudson, 2005; Le Gall et ak, 2004; Reff & Heard, 2001 ; Reiter et ak , 1996; and Y oung et ak , 1995 further describe and enable the production of effective antibody fragments. In some embodiments, the antibody is a “chimeric” antibody as described in U.S. Pat. No. 4,816,567. In some embodiments, the antibody is a humanized antibody, such as described U.S. Pat. Nos. 6,982,321 and 7,087,409. In some embodiments, the antibody is a human antibody. A “human antibody” such as described in US 6,075,181 and 6,150,584. In some embodiments, the antibody is a single domain antibody such as described in EP 0368 684, WO 06/030220 and WO 06/003388.

In a particular embodiment, the sialidase inhibitor activity is a monoclonal antibody. Monoclonal antibodies 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, the human B-cell hybridoma technique and the EBV-hybridoma technique.

In a particular, the inhibitor is an intrabody having specificity for sialidase. As used herein, the term "intrabody" generally refer to an intracellular antibody or antibody fragment. Antibodies, in particular single chain variable antibody fragments (scFv), can be modified for intracellular localization. Such modification may entail for example, the fusion to a stable intracellular protein, such as, e.g., maltose binding protein, or the addition of intracellular trafficking/localization peptide sequences, such as, e.g., the endoplasmic reticulum retention. In some embodiments, the intrabody is a single domain antibody. In some embodiments, the antibody according to the invention is a single domain antibody. The term “single domain antibody” (sdAb) or "VHH" refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called “nanobody®”. According to the invention, sdAb can particularly be llama sdAb.

In some embodiments, the inhibitor of sialidase activity is a small organic molecule.

The term "small organic molecule" as used herein, refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macro molecules (e. g. proteins, nucleic acids, etc.). Typically, small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.

In a particular embodiment, the sialidase inhibitor includes, but is not limited to one or more of the following: fetuin, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA) or a pharmaceutically acceptable salt thereof; Oseltamivir (ethyl (3R,4R,5S)-5-amino-4-acetamido- 3-(pentan-3-yloxy)-cyclohex-l-ene-l-carboxylate); Zanamivir ((2R,3R,4S)-4-guanidino-3- (prop-l-en-2-ylamino)-2-((lR,2R)-l,2,3-trihydroxypropyl)-3,4-dihydro-2H-pyran-6- carboxylic acid); Laninamivir ((4S,5R,6R)-5-acetamido-4-carbamimidamido-6-[(lR,2R)-3- hydroxy-2-methoxypropyl]-5,6-dihydro-4H-pyran-2-carboxylic acid); and Peramivir ((lS,2S,3S,4R)-3-[(lS)-l-acetamido-2-ethyl-butyl]-4-(diaminomethylideneamino)-2- hydroxy-cyclopentane- 1 -carboxylic acid) or a pharmaceutically acceptable salt thereof.

In another embodiment, the sialidase inhibitor is the sodium salt of 2,3-dehydro-2- deoxy-N-acetylneuraminic acid or a combination thereof. Sialidase inhibitors used with the present invention include those known in the art or those later developed.

In a particular embodiment, the sialidase inhibitor is Oseltamivir or a variant thereof also known as ethyl (3R,4R,5S)-5-amino-4-acetamido-3-(pentan-3-yloxy)-cyclohex-l-ene-l- carboxylate (Tamiflu, Genentech, Cambridge, Mass.) Oseltamivir has the following Cas number in the art: 196618-13-0.

In a particular embodiment, the sialidase inhibitor is Zanamivir or a variant thereof also known as ((2R,3R,4S)-4-guanidino-3-(prop-l-en-2-ylamino)-2-((lR,2R)-l,2,3- trihydroxypropyl)-3,4-dihydro-2H-pyran-6-carboxylic acid) (Relenza; Glaxo Smith Kline, Research Triangle Park, N.C.). Zanamivir has the following Cas number in the art: 139110-80- 8

In a particular embodiment, the sialidase inhibitor is Peramivir or a variant thereof ((lS,2S,3S,4R)-3-[(lS)-l-acetamido-2-ethyl-butyl]-4-(diaminomethylideneamino)-2- hydroxy-cyclopentane- 1 -carboxylic acid) (BioCry st, Birmingham, Ala.). Peramivir has the following Cas number in the art: 229614-55-5.

In a particular embodiment, the sialidase inhibitor is Laninamivir or a variant thereof also known as (2R,3R,4S)-3-acetamido-4-(diaminomethylideneamino)-2-[(lR,2R)-2,3- dihydroxy-l-methoxypropyl]-3,4-dihydro-2H-pyran-6-carboxylic acid (marketed under the name Inavir by Daiichi Sankyo). It is currently in Phase III clinical trials Laninamivir has the following Cas number in the art: 203120-17-6. Small molecules as described above may also be derivatized, for example, bearing modifications other than insertion, deletion, or substitution of amino acid residues, thus resulting in a variation of the original product (a variant). These modifications can be covalent in nature, and include for example, chemical bonding with lipids, other organic moieties, inorganic moieties, and polymers. For reviews on viral sialidase/neuraminidase inhibitors, please see “The war against influenza: discovery and development of sialidase inhibitors.” Nature Reviews: Drug Discovery (2007) 6 (12): 967-74. Klumpp et al., (2006) Curr. Top. Med. Chem. 6(5):423-34; Zhang et al., (2006) Mini Rev. Med. Chem. 6(4):429-48; Jefferson et al., (2006) Lancet 367(9507):303-13; Alymova et al., (2005) Curr Drug Targets Infect. Disord. 5 (4): 401-9; Moscona (2005) N. Engl. J. Med. 353(13): 1363-73; De Clercq (2004) J. Clin. Virol. 30(2): 115-33; Stiver (2003) CMAJ 168(l):49-56; Oxford et al., (2003) Expert Rev. Anti. Infect. Ther. l(2):337-42; Cheer et al., (2002) Am. J. Respir. Med. 1(2): 147-52; Sidewell et al., (2002) Expert Opin. Investig. Drugs. 11(6): 859-69; Doucette et al., (2001) Expert Opin. Pharmacother. 2(10): 1671-83; Young et al., (2001) Philos. Trans. R. Soc. Lond. B. Biol. Sci. 356(1416): 1905- 13; Lew etal., (2000) Curr. Med. Chem. 7(6):663-72); Taylor et al., (1996) Curr. Opin. Struct. Biol. 1996 6(6): 830-7 and published U.S. Patent Application. Nos. 2009/0175805, 2006/0057658, 2008/0199845 and 2004/0062801, the entirety of each of which is incorporated herein by reference.

In a particular embodiment, the method according to the invention, wherein the sialidase inhibitor gene expression is siRNA, shRNA, miRNA, antisense oligonucleotide, or a ribozyme.

In some embodiments, the sialidase inhibitor expression is an antisense oligonucleotide. Anti-sense oligonucleotides, including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of sialidase mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of sialidase proteins, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding sialidase can be synthesized, e.g., by conventional phosphodiester techniques and administered by e.g., intravenous, or subcutaneous injection. Methods for using antisense techniques for specifically alleviating gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732).

In a particular embodiment, the sialidase inhibitor expression is a shRNA. shRNA is generally expressed using a vector introduced into cells, wherein the vector utilizes the U6 promoter to ensure that the shRNA is always expressed. This vector is usually passed on to daughter cells, allowing the gene silencing to be inherited. The shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs that match the siRNA to which it is bound.

In some embodiments, the sialidase inhibitor expression is a small inhibitory RNAs (siRNAs). Sialidase expression can be reduced by contacting the subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that ialidase 1 expression is specifically inhibited (i.e. RNA interference or RNAi). Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g. see Tuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ. (2002); McManus, MT. et al. (2002); Brummelkamp, TR. et al. (2002); U.S. Pat. Nos. 6,573,099 and 6,506,559; and International Patent Publication Nos. WO 01/36646, WO 99/32619, and WO 01/68836). In a particular embodiment, the siRNA is ALN-PCS02 developed by Alnylam (phase 1 ongoing).

In some embodiments, the sialidase inhibitor expression is a ribozyme. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of sialidase mRNA sequences are thereby useful within the scope of the present invention. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays.

In some embodiments, the sialidase inhibitor expression is an endonuclease. The term “endonuclease” refers to enzymes that cleave the phosphodiester bond within a polynucleotide chain. Some, such as Deoxyribonuclease I, cut DNA relatively nonspecifically (without regard to sequence), while many, typically called restriction endonucleases or restriction enzymes, and cleave only at very specific nucleotide sequences. The mechanism behind endonuclease-based genome inactivating generally requires a first step of DNA single or double strand break, which can then trigger two distinct cellular mechanisms for DNA repair, which can be exploited for DNA inactivating: the errorprone nonhomologous end-joining (NHEJ) and the high-fidelity homology-directed repair (HDR). In a particular embodiment, the endonuclease is CRISPR- cas. As used herein, the term “CRISPR-cas” has its general meaning in the art and refers to clustered regularly interspaced short palindromic repeats associated which are the segments of prokaryotic DNA containing short repetitions of base sequences. In some embodiment, the endonuclease is CRISPR-cas9 which is from Streptococcus pyogenes. The CRISPR/Cas9 system has been described in US 8697359 B1 and US 2014/0068797. In some embodiment, the endonuclease is CRISPR-Cpfl which is the more recently characterized CRISPR from Provotella and Francisella 1 (Cpfl) in Zetsche et al. (“Cpfl is a Single RNA-guided Endonuclease of a Class 2 CRISPR-Cas System (2015); Cell; 163, 1-13).

In a sixth aspect, the invention relates to a method for treating a subject suffering from a platelet desialylation related disease, comprising the steps of: i) calculating the ratio between RCA and FSC as described above and ii) administering said subject a sialidase inhibitor when the platelets are considered as desialylated according to the invention.

Typically, the invention relates to the invention relates to a method for treating a subject suffering from a platelet desialylation related disease, comprising the steps of: i) identifying non-specific populations appearing within the platelet population; ii) determining the concentration of RCA and the value of FSC in said biological sample; iii) calculating the ratio between RCA and FSC; iv) comparing said ratio with a range; and v) administering said subject a sialidase inhibitor when the platelets are considered as desialylated according to the invention.

As used herein, the terms "treating" or "treatment" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subject at risk of contracting the disease or suspected to have contracted the disease as well as subject who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).

In a particular embodiment, the subject is a human who is susceptible to have an influenza virus A infection. In a particular embodiment, the subject is a human who is susceptible to have disorder related to Trans Aortic Valve Implantation (TAVI) or Mechanical Circulatory Support Implantation (MCSI).

In a particular embodiment, the sialidase inhibitors are described above.

Typically, when the ratio calculated is in the reference range (between 0.237 and 0.941), it means that subject will achieve a response with a sialidase inhibitor and thus the method according to the invention is suitable to treat said subject with a sialidase inhibitor.

Typically, when the ratio is outside the reference range (inferior to 0.237 or superior to 0.941), it means that the subject will not achieve a response with a sialidase inhibitor and thus the method according to the invention is not suitable to treat said subject with a sialidase inhibitor.

The inhibitors of sialidase as described above may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions.

Accordingly, in a seventh aspect, the invention relates to a pharmaceutical composition comprising inhibitors of sialidase and pharmaceutically acceptable excipients.

As used herein, the terms "pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to anon-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The polypeptide (or nucleic acid encoding thereof) can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or inj ected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

In an eighth aspect, the invention a kit for performing the method according to the invention, wherein said kit comprises i) means for identifying non-specific populations appearing within the platelet population (for example with a Mitotracker), ii) means for determining RCA and FSC in a biological sample obtained from a subject; and iii) instructions notice with a reference range determined in a healthy population as described above. In a further embodiment, the kit according to the invention allows to measure the concentration or quality of RCA in lyophilized platelets.

In some embodiment, the means for identifying non-specific populations appearing within the platelet population is a Mitotracker, such as MitoTracker™ Deep Red FM.

In some embodiment, the means for determining RCA in a biological sample is FITC- conjugated RCA.

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.

FIGURES:

Figure 1: RCA labeling, concentrations and specificity

A) A flow cytometry analysis of RCA binding on PRP containing various concentrations of RCA (1.56, 3.12, 6.25, and 12.5 pg/mL) (n=3 experiments). B) A histogram of the flow cytometry analysis of RCA binding in PRP treated (or not) with 0.5 U/mL NeuC. C) A histogram of the flow cytometric analysis of RCA binding in PRP incubated (or not) with 200 mM b-lactose (n=4 experiments). The mean ± SD values were compared using Student’s t-test.

Figure 2: Effect of the platelet count on RCA labeling

A flow cytometry analysis of RCA binding on PRP at indicated platelet concentration (n=3 experiments). The mean ± SD values were compared using a one-way ANOVA and Dunnett’s post test.

Figure 3: Influence of the time interval on RCA binding, glycoprotein expression and FSC values

A flow cytometry analysis of (A) RCA binding, (B) FSC parameter, (C) anti-GPIba binding and (D) anti-GPIIb binding in PRP at the indicated time points. Analyses performed at 30, 60, 120, 180 and 240 min after the incubation had been stopped with PBS (n=3 experiments), and the RCA/size parameter ratios were calculated: (E) RCA/FSC, (F) RCA/GPIba and (G) RCA/GPIIb. The mean ± SD values were compared using a one-way ANOVA and Dunnett’s post test.

Figure 4: Influence of storage of whole blood for 24 hours at room temperature

Samples of whole blood were stored at room temperature (22°C) for 24 hours (Day 1, Dl) after collection (Day 0, DO). Dot plots of a flow cytometry analysis of (A) RCA binding, (B) anti-GPIIb binding, (C) anti-GPIba binding and (D) FSC in PRP (n=10 experiments). The mean ± SD values were compared using Student’s t-test. (E) Dot plots of the RCA/FSC ratio on DO and Dl.

Figure 5: Values of RCA, FSC and RCA/FSC in healthy subjects

Dot plots of a flow cytometry analysis of (A) RCA binding and (D) FSC in 127 healthy subjects and compared (B-E) for males (n=49) vs. females (n=68) and (C-F) among blood groups (O: n=39; A: n=45; B: n=13; AB: n=4). The RCA/FSC ratio was calculated, and outliers were removed. Dot plots of the RCA/FSC ratio (G) for 120 healthy subjects. The RCA/FSC ratio was compared (H) in males (n=44) vs. females (n=67) and (I) among blood groups (O: n=38; A: n=44; B: n=12; AB: n=4). The statistical analyses are described in the Material and Methods section. RCA, FSC, and RCA/FSC values for males and females were compared using a Mann-Whitney test. RCA, FSC, and RCA/FSC values for blood groups were compared using a one-way ANOVA and Dunnett’s post test.

Figure 6: Platelets staining with mitotracker. (A) Platelets labeling with mitotracker at different concentrations (25, 100, 250, 500 mM), the signal was measured in the FL4-H channel of the flow cytometer. The dashed line indicated the signal of red blood cells (RBC) in the presence of mitotracker, as RBC are anucleated, this signal corresponded to the background. (B) Mitotracker (100 nM) was used in thrombocytopenic patients (without disease classification). The % of events (i), and the RCA (ii) were measured in the platelet gate with and without mitotracker.

Figure 7: Variation of RCA staining on lyophilized platelets. Platelets were resuspended at a concentration of 150 G/L in PBS and then diluted at 50G/L in the assay. Platelets were then stained with the RCA-FITC at 12.5 pg/niL. Two vials of RCA-FITC from the same lot were then used with a delay of opening of 12 months. Each vial was referred as old (opened 12 months ago) and young vial (recently opened).

EXAMPLES:

Example 1:

Material & Methods

Study population

Blood samples from healthy subjects were obtained from the French Blood Establishment (Etablissement Francais du Sang, Paris, France; reference: 13/CABANEL/008), in accordance with the tenets of the Declaration of Helsinki, the study participants were informed about the anonymous use of their personal data, and gave their written, informed consent. In compliance with the Clinical and Laboratory Standards Institute (CLSI) C28A3 guideline [26], reference ranges were established by testing 127 healthy subjects, all of whom had not taken any anti-platelet medications for at least 2 weeks prior to blood sampling.

Blood sampling and preparation

In line with international guidelines, venous blood was collected in vacutainer® containing 7.5% K3 EDTA [27] Platelet-rich plasma (PRP) was obtained after two centrifugation steps, i.e. 10 minutes at 38 g and then 10 minutes at 122 g, to enable the recovery of large and giant platelets.

Flow cytometry

Platelet surface b-galactose exposure was determined using FITC-conjugated RCA (Vector Labs, Burlingame, CA). Briefly, PRP diluted 1/20 with PBS (95 pL) was incubated with RCA for 30 min at room temperature in a final volume of 100 pL. The reaction was stopped with PBS (400 pL) and then analyzed using flow cytometry. In some experiments, samples in which RCA had been incubated with b-lactose (200 mM) (Sigma-Aldrich, Saint- Quentin Fallavier, France) were used as negative controls [25, 28] As a positive control of platelet desialylation (i.e. the maximum possible amount of b-galactose exposure due to sialic acid removal from the platelet surface), PRP was incubated with 0.5 U/mL neuraminidase from Clostridium perfringens (NeuC, Sigma-Aldrich) for 20 minutes at 37°C. The stability of platelet b-galactose exposure was studied from 10 controls. On the day of sampling, each blood sample was divided into two aliquots; one was assayed in the two hours, and the other was assayed after 24 hours of storage at room temperature. The stability of RCA binding was evaluated 30 minutes and 1, 2, 3 and 4 hours after the start of the incubation.

Surface GPIba and GPIIb expression was measured with mouse anti-GPIba (clone SZ2) or anti-GPIIb (clone P2) antibodies, respectively, or a negative isotype control (Biocytex, Marseille, France), and detected with a FITC-labeled secondary antibody. After 30 minutes of incubation at room temperature, samples were diluted with PBS and analyzed using flow cytometry.

Lectin or antibody binding was determined on a FACS CANTO-II™ flow cytometer, and the resulting data were analyzed using Diva 6 software (both from BD Biosciences, San Jose, CA). The flow cytometer were routinely calibrated with SPHERO™ Rainbow Calibration Particles (BD Biosciences, Le Pont de Claix, France). In each sample, the geometric mean fluorescence intensity (MFI) was determined for a total of 5,000 platelets. Statistical analysis

Quantitative data were graphed as dot plots (showing the mean ± standard deviation (SD)) or as box-and-whisker plots (with the lower and upper hinge defining the interquartile range, the line inside the box denoting the median, and the whiskers corresponding to the maximum and the minimum). All statistical analyses were performed with Prism 6 for Mac software (version 6; GraphPad, Inc., San Diego, CA). If only two groups were compared, Student’s t-test (for data with a Gaussian distribution, according to a D’Agostino-Person test) or Mann-Whitney test (for data with a non-Gaussian distribution) was used. For three or more groups, a one-way analysis of variance (ANOVA) and Dunnetfs correction for multiple comparisons were applied. Equality of variance was tested with an F test (prior to Student’s t- test) or Bartlett’s test (prior to an ANOVA). The assay’s standardized results are presented as mean ± SD. In accordance with CLSI guideline C28-A3 [26], the reference range was defined as the 95% confidence interval (Cl) for the population. Correlations were assessed by calculating Pearson’s coefficient r. The threshold for statistical significance was set to p<0.05 (*p<0.05; **p<0.01; ***p<0.001). To remove outliers, we applied the robust regression and outlier removal (ROUT) method [29], with a Q coefficient of 1%.

Results

RCA binding: concentrations and specificity

As lectins are known to induce hemagglutination when incubated with whole blood [30], we decided to study platelet b-galactose exposure in PRP. Furthermore, and given that thrombocytopenic patients’ blood samples are often collected in EDTA tubes, we first used flow cytometry to evaluate the binding of RCA to platelets in EDTA-containing PRP and thus determined the optimal concentration of RCA. Hence, PRP was incubated with FITC-labeled RCA concentrations ranging from 0 to 50 pg/mL (Figure 1A and data not shwon). RCA binding to platelets was observed at all concentrations. Between 1.56 to 12.5 pg/mL, the level of RCA binding increased in a linear manner (Figure 1A). In contrast, RCA data > 25 pg/mL were not usable. In fact, the flow cytometry histogram for FITC showed a large peak at 25 pg/mL in the last decade of the FL1-H filter (data not shwon) and for 50 pg/mL RCA, a non specific platelet population (P2, corresponding to platelet agglutination) was found outside the platelet gate (i.e. outside the forward scatter (FSC)/SSC window) (data not shwon). The concentration of 12.5 pg/mL RCA represented a good compromise with no agglutination observed, and a FITC-MFI peak of 2147 ± 838 (n=3).

We next evaluated the maximum possible levels of b-galactose exposure on platelets (corresponding to a positive control) by incubating PRP with 0.5 U/mL NeuC prior to incubation with 12.5 mg/mL RCA (n=3); we found that the MFI for RCA labeling was 8.4 ± 2.4 fold higher than in control PRP (Figure IB). At higher RCA concentrations (>25 pg/mL). all flow cytometry measurement of RCA binding in the presence of NeuC were out the range (data not shwon). These results demonstrated that an RCA concentration of 12.5 pg/mL is not a limiting factor and that a strong desialylation (i.e. an increase in the MFI) can easily be observed, relative to a control.

To study the specificity of our assay, we performed a competition experiment with 200 mM b-lactose. As a disaccharide composed of galactose and glucose, b-lactose can compete for RCA binding with the b-galactose on glycoproteins exposed after terminal sialic acid removal [25, 28] As expected, we observed a shift in the signal and thus a strong decrease in RCA binding to platelets (RCA MFI: 248 ± 98 for RCA + b-lactose, and 1805 ± 542 for RCA alone) (Figure 1C), attesting the assay’s specificity. With 12.5 pg/mL RCA (10 measurements in 4 healthy subjects), the intra-assay coefficient of variation (CV) ranged from 7% to 12%. Under these conditions, the mean ± SD RCA MFI for fixed platelets was 8128 ± 960 (n=15) and the inter-assay CV was 11.8% (data not shwon).

Taken as a whole, our data demonstrated that platelet sialylation was measurable with 12.5 pg/mL RCA. This concentration yielded an acceptable signal -to-noise ratio, and enabled the detection of an eight-fold increase in platelet b-galactose exposure (Figure IB).

Standardization of the RCA-based assay: influence of the platelet count, RCA binding stability, platelet size, and sample storage conditions

We next sought to determine whether the platelet count, stability of the RCA binding, platelet size, platelet storage conditions, had any influenced on the assay results.

Platelet count. We first analyzed the effect of the platelet count by comparison of MFI- RCA from 3 healthy subjects before adjustment of the PRP’s platelet count (525 ± 52 xlO⁹ L ') and after adjustment (100, 50, 20 and 10 xlO⁹ L ¹). There were no significant differences in the MFI-RCA between the various dilutions; the MFI-RCA was 1109 ± 233 with a platelet count of 10 xlO⁹ L ¹ and 874 ± 208 in the unadjusted PRP (Figure 2). These data demonstrate that a platelet count as low as 10 xlO⁹ L ¹ does not induce a variation with a RCA concentration of 12.5 pg/mL.

RCA binding stability. We next evaluated the influence of the time interval between the moment when lectin association was stopped with PBS and the flow cytometry measurement (30, 60, 120, 180 or 240 min). The RCA binding was stable up to 240 min (MFI- RCA: 1233 ± 329 at To vs 1480±410 at T₂₄o: p=NS) (Figure 3A). Platelet size. As we have previously reported, the possible presence of large and/or giant platelets means that the MFI for RCA must be normalized against platelet size [9, 25] On the day when the blood sample was collected, we measured the FSC (the amount of laser light that passes around the cell, and is proportional to the cell’s diameter) and the levels of two conventional glycoproteins (GPIba and GPIIb) proportional with the mean platelet volume [31], at the same time as RCA binding. The FSC parameter and the two glycoprotein levels were stable up for to 240 min after the incubation had been stopped (Figure 3B-D). The RCA/size ratios (RCA/FSC, RCA/GPIba and RCA/GPIIb were stable over the 4 hours of measurement (Figure 3E-G). These data show that if the assay is performed on the day when the blood sample is collected, the MFI for RCA can be normalized against FSC, GPIba and/or GPIIb for up to 4 hours.

Sample storage. We next investigated the effect of the sample storage time prior to RCA binding. It has been proven that chilled platelets undergo desialylation [32], indeed the MFI-RCA of blood stored at 4°C was 1939 ± 441 compared to control 1140 ± 297 (p<0.001). Whole blood was therefore stored at room temperature (22°C) for 24h (Dl) after sample collection (DO). We first compared the MFI-RCA values on DO and Dl ; interestingly, the values were similar (1252 ± 434 and 1140 ± 297, respectively, p>0.05) (Figure 4A). In contrast, levels of both of GPIIb and GPIba on Dl differed from those observed on DO (Figure 4B-C). The MFI for GPIIb was 11548 ± 3172 on DO and 5963 ± 2155 on Dl (p O.OOl), whereas the MFI for GPIba was 7033 ± 740 on DO and 8040 ± 1006 on Dl (p<0.05). However, the FSC parameter was the same on DO and Dl (Figure 4D); hence, the RCA/FSC ratio was also the same on DO and Dl (Figure 4E).

Taken as a whole, our data show that platelet b-galactose exposure can be measured in an RCA-based flow cytometry assay of a PRP sample stored at room temperature for up to 24 hours. The measure of RCA binding did not change significantly over the first 4 hours of incubation. To take account of platelet size, we selected the FSC parameter. Hence, platelet b- galactose exposure was expressed as an RCA/FSC ratio (Table 1).

Reference values of platelet b-galactose exposure (RCA/FSC ratio) in healthy subjects

After having defined standardized conditions for measuring b-galactose exposure at the platelet surface, we measured the variability of platelet b-galactose exposure (RCA binding), FSC and the RCA/FSC ratio in a population of 127 healthy subjects. We recorded the donors’ sex and blood group, and checked whether these variables had an influence on the RCA, FSC or RCA/FSC ratio. The sex was known for 117 donors (49 men and 68 women), and blood group data were confirmed for 101 donors (Table 2). The hemoglobin level, leukocyte count, platelet count, and mean platelet volume were within the reference ranges [33] for this population (Table 2). The age median was 28. In this population of healthy subjects, the RCA values were not normally distributed. The median [95%CI] MFI-RCA was 1232 [652-2874] (Figure 5A, Table 3). As expected in a population of healthy subjects, the MFI-RCA was not correlated with the platelet count or the FSC variable (data not shown). Interestingly, there were no differences between men and women or between ABO blood groups (Figure 5B-C). The median [95%CI] FSC was 2310 [1282-3894] (Figure 5D, Table 3). Again, there were no differences with regard to sex or ABO blood group (Figure 5E-F). We next evaluated the variability of the RCA/FSC ratio. After rejecting outliers (7 outliers have been identified), we determined reference values for 120 healthy subjects. We found that the RCA/FSC values were not normally distributed. The median [95%CI] RCA/FSC ratio was 0.52 [0.237-0.941] (Figure 5G, Table 3). Interestingly, the RCA/FSC ratio was not different according to the sex (median RCA/FSC ratio: 0.49 for women and 0.52 for men) (Figure 5H). Similarly, the RCA/FSC ratio did not vary significantly with the blood group (Figure 51) - meaning that matching for these two parameters will not be required in a future clinical trial. Taken as a whole, we found that the RCA/FSC ratio in a population of healthy subjects was independent of sex and blood group, and has a reference range ([95%CI] of 0.237 to 0.941. Table 1: Technical and biological parameters for platelet b-galactose measurement with RCA lectin by flow cytometry

Table 2: Biological data of the cohort

Table 3: Statistic values of RCA, FSC and RCA/FSC ratio in the healthy population.

Example 2: Platelets staining with mitotracker

Mitochondrion in platelets were stained with the mitotracker (MitoTracker™ Deep Red FM, Thermo Fischer Scientific, numiro de catalogue: M22426 . In a first experiment the optimal concentration was investigated between 500, 25(1 100 et 25 nM. Briefly, PRP diluted 1/20 with PBS (95 pL) was incubated with Mitotracker for 30 min at room temperature in a final volume of 100 pL. The reaction was stopped with PBS (400 pL) and then analyzed using flow cytometry (Accuri BD).

Results: Based on the figure 6A, we choose the concentration of 100 nM to detect platelets positive for the mitotracker.

Results: The % of events measured without mitotracker was 90 ± 15%, when mitotracker was added the number of events was 52 ± 16%. This data suggested that in thrombocytopenic PRP, a non-specific population (without mitochondrion) was present. The RCA values (MFI) were 21279 ±8626 without mitotracker and 9178 ± 3546 in the presence of mitotracker (ii). These data demonstrated that using mitotracker allow to remove non-specific population that are RCA positive. The RCA values were therefore refined.

Conclusion: in thrombocytopenic patients a double staining with a first identification of platelets with mitotracker is necessary before to measure the RCA values. The RCA values have to be measured in a double population mitotracker +/RCA+. Example 3: Measure of the RCA quality

To ensure that the RCA is usable (giving the same signal of fluorescence from the opening of the vial), we used fixed lyophilized platelets that are desialylated (from Siemens Healthcare Diagnostics, Saint-Denis, France). Platelets were resuspended at a concentration of 150 G/L in PBS and then diluted at 50G/L in the assay. Platelets were then stained with the RCA-FITC at 12.5 pg/mL. Two vials of RCA-FITC from the same lot were then used with a delay of opening of 12 months. Each vial was referred as old (opened 12 months ago) and young vial (recently opened).

Results: The signal of RCA-FITC decreased by 2.5 fold times after 12 months of storage.

Conclusion: Fixed lyophilized platelets could be used as quality control of RCA. A decrease of < 10% of the MFI of the RCA-FITC of same RCA vial has to be considered to rule out the vial.

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.

1. Fabris F, Cordiano I, Steffan A, Ramon R, Scandellari R, Nichol JL, et al. Indirect study of thrombopoiesis (TPO, reticulated platelets, glycocalicin) in patients with hereditary macrothrombocytopenia. Eur J Haematol. 2000 Mar;64(3):151-6. PubMed PMID: 10997880.

2. Sorensen AL, Rumjantseva V, Nayeb-Hashemi S, Clausen H, Hartwig JH, Wandall HH, et al. Role of sialic acid for platelet life span: exposure of beta-galactose results in the rapid clearance of platelets from the circulation by asialoglycoprotein receptor-expressing liver macrophages and hepatocytes. Blood. 2009 Aug 20;114(8): 1645-54. PubMed PMID: 19520807. Pubmed Central PMCID: PMC2731641.

3. Hoffmeister KM. The role of lectins and glycans in platelet clearance. Journal of thrombosis and haemostasis : JTH. 2011 Jul;9 Suppl 1:35-43. PubMed PMID: 21781240. Pubmed Central PMCID: 4303235.

4. Grozovsky R, Begonja AJ, Liu K, Visner G, Hartwig JH, Falet H, et al. The Ashwell-Morell receptor regulates hepatic thrombopoietin production via JAK2-STAT3 signaling. Nature medicine. 2015 Jan;21(l):47-54. PubMed PMID: 25485912. Pubmed Central PMCID: 4303234. 5. Li Y, Fu J, Ling Y, Yago T, McDaniel JM, Song J, et al. Sialylation on O-glycans protects platelets from clearance by liver Kupffer cells. Proceedings of the National Academy of Sciences of the United States of America. 2017 Aug 1 ; 114(31): 8360-5. PubMed PMID: 28716912. Pubmed Central PMCID: PMC5547648.

6. Deppermann C, Kratofil RM, Peiseler M, David BA, Zindel J, Castanheira F, et al. Macrophage galactose lectin is critical for Kupffer cells to clear aged platelets. The Journal of experimental medicine. 2020 Apr 6;217(4). PubMed PMID: 31978220.

7. Futterer J, Dalby A, Lowe GC, Johnson B, Simpson MA, Motwani J, et al. Mutation in GNE is associated with a severe form of congenital thrombocytopenia. Blood. 2018 Jun 25. PubMed PMID: 29941673.

8. Izumi R, Niihori T, Suzuki N, Sasahara Y, Rikiishi T, Nishiyama A, et al. GNE myopathy associated with congenital thrombocytopenia: a report of two siblings. Neuromuscular disorders : NMD. 2014 Dec;24(12): 1068-72. PubMed PMID: 25257349.

9. Kauskot A, Pascreau T, Adam F, Bruneel A, Reperant C, Lourenco-Rodrigues MD, et al. A mutation in the gene coding for the sialic acid transporter SLC35A1 is required for platelet life span but not proplatelet formation. Haematologica. 2018 Aug 16. PubMed PMID: 30115659.

10. Mohamed M, Ashikov A, Guillard M, Robben JH, Schmidt S, van den Heuvel B, et al. Intellectual disability and bleeding diathesis due to deficient CMP— sialic acid transport. Neurology. 2013 Aug 13;81(7):681-7. PubMed PMID: 23873973.

11. Ng BG, Asteggiano CG, Kircher M, Buckingham KJ, Raymond K, Nickerson DA, et al. Encephalopathy caused by novel mutations in the CMP-sialic acid transporter, SLC35A1. Am J Med Genet A. 2017 Nov;173(ll):2906-ll. PubMed PMID: 28856833. Pubmed Central PMCID: PMC5650519.

12. Revel-Vilk S, Shai E, Turro E, Jahshan N, Hi-Am E, Spectre G, et al. GNE variants causing autosomal recessive macrothrombocytopenia without associated muscle wasting. Blood. 2018 Oct 25;132(17):1851-4. PubMed PMID: 30171045. Pubmed Central PMCID: 6202914.

13. Willig TB, Breton-Gorius J, Elbim C, Mignotte V, Kaplan C, Mollicone R, et al. Macrothrombocytopenia with abnormal demarcation membranes in megakaryocytes and neutropenia with a complete lack of sialyl-Lewis-X antigen in leukocytes-a new syndrome? Blood. 2001 Feb l;97(3):826-8. PubMed PMID: 11157507.

14. Li MF, Li XL, Fan KL, Yu YY, Gong J, Geng SY, et al. Platelet desialylation is a novel mechanism and a therapeutic target in thrombocytopenia during sepsis: an open-label, multicenter, randomized controlled trial. J Hematol Oncol. 2017 May 11 ;10(1): 104. PubMed PMID: 28494777. Pubmed Central PMCID: 5426054.

15. Li J, Callum JL, Lin Y, Zhou Y, Zhu G, Ni H. Severe platelet desialylation in a patient with glycoprotein Ib/IX antibody-mediated immune thrombocytopenia and fatal pulmonary hemorrhage. Haematologica. 2014 Apr;99(4):e61-3. PubMed PMID: 24532041. Pubmed Central PMCID: 3971097.

16. Li J, van der Wal DE, Zhu G, Xu M, Yougbare I, Ma L, et al. Desialylation is a mechanism of Fc-independent platelet clearance and a therapeutic target in immune thrombocytopenia. Nature communications. 2015;6:7737. PubMed PMID: 26185093. Pubmed Central PMCID: 4518313.

17. Shao L, Wu Y, Zhou H, Qin P, Ni H, Peng J, et al. Successful treatment with oseltamivir phosphate in a patient with chronic immune thrombocytopenia positive for anti- GPIb/IX autoantibody. Platelets. 2015;26(5):495-7. PubMed PMID: 25166956.

18. Tao L, Zeng Q, Li J, Xu M, Wang J, Pan Y, et al. Platelet desialylation correlates with efficacy of first-line therapies for immune thrombocytopenia. J Hematol Oncol. 2017 Feb 08;10(1):46. PubMed PMID: 28179000. Pubmed Central PMCID: PMC5304552.

19. Alioglu B, Tasar A, Ozen C, Selver B, Dallar Y. An experience of oseltamivir phosphate (tamiflu) in a pediatric patient with chronic idiopathic thrombocytopenic purpura: a case report. Pathophysiol Haemost Thromb. 2010;37(2-4):55-8. PubMed PMID: 21063076.

20. Zhang XH, Wang QM, Zhang JM, Feng FE, Wang FR, Chen H, et al. Desialylation is associated with apoptosis and phagocytosis of platelets in patients with prolonged isolated thrombocytopenia after allo-HSCT. J Hematol Oncol. 2015;8: 116. PubMed PMID: 26497387. Pubmed Central PMCID: PMC4619537.

21. Marini I, Zlamal J, Faul C, Holzer U, Hammer S, Pelzl L, et al. Autoantibody- mediated desialylation impairs human thrombopoiesis and platelet life span. Haematologica. 2019 Dec 19. PubMed PMID: 31857361.

22. Zheng SS, Perdomo JS, Leung HHL, Yan F, Chong BH. Acquired Glanzmann thrombasthenia associated with platelet desialylation. Journal of thrombosis and haemostasis : JTH. 2020 Mar; 18(3): 714-21. PubMed PMID: 31869497.

23. Revilla N, Corral J, Minano A, Mingot-Castellano ME, Campos RM, Velasco F, et al. Multirefractory primary immune thrombocytopenia; targeting the decreased sialic acid content. Platelets. 2018 Oct 8:1-9. PubMed PMID: 30296193. 24. Deng W, Xu Y, Chen W, Paul DS, Syed AK, Dragovich MA, et al. Platelet clearance via shear-induced unfolding of a membrane mechanoreceptor. Nature communications. 2016 Sep 27;7: 12863. PubMed PMID: 27670775.

25. Dupont A, Soukaseum C, Cheptou M, Adam F, Nipoti T, Lourenco-Rodrigues MD, et al. Relevance of platelet desialylation and thrombocytopenia in type 2B von Willebrand disease: preclinical and clinical evidence. Haematologica. 2019 Dec;104(12):2493-500. PubMed PMID: 30819911. Pubmed Central PMCID: 6959185.

26. Horowitz M, Altaie S, Boyd J, Ceriotti F, Garg U, Horn P, et al. CLSI Document C28-A3. Defining, establishing, and verifying references intervals in the clinical laboratory; approved guideline. 28. Wayne, Pa.: Clinical and Laboratory Standards Institute; 2010. p. volumes.

27. WHO Expert Committee on Biological Standardization: Geneva -o. 2002. p.

107.

28. Drysdale RG, Herrick PR, Franks D. The specificity of the haemagglutinin of the Castor bean, Ricinus communis. Vox Sang. 1968; 15(3): 194-202. PubMed PMID: 5693226.

29. Motulsky HJ, Brown RE. Detecting outliers when fitting data with nonlinear regression - a new method based on robust nonlinear regression and the false discovery rate. BMC bioinformatics. 2006 Mar 9;7:123. PubMed PMID: 16526949. Pubmed Central PMCID: 1472692.

30. Adamova L, Mabnovska L, Wimmerova M. New sensitive detection method for lectin hemagglutination using microscopy. Microscopy research and technique. 2014 Oct;77(10):841-9. PubMed PMID: 25060388.

31. Yakushkin VV, Zyuryaev IT, Khaspekova SG, Sirotkina OV, Ruda MY, Mazurov AV. Glycoprotein Ilb-IIIa content and platelet aggregation in healthy volunteers and patients with acute coronary syndrome. Platelets. 2011;22(4):243-51. PubMed PMID: 21329420.

32. Hoffmeister KM, Felbinger TW, Falet H, Denis CV, Bergmeier W, Mayadas TN, et al. The clearance mechanism of chilled blood platelets. Cell. 2003 Jan 10; 112(1): 87-97. PubMed PMID: 12526796.

33. Troussard X, Vol S, Comet E, Bardet V, Couaillac JP, Fossat C, et al. [Determination of full blood count normal reference values for adults in France] Annales de biologie clinique. 2014 Sep-Oct;72(5):561-81. PubMed PMID: 25336130. Etude des valeurs normales de l'hemogramme chez l'adulte: un besoin pour une meilleure interpretation et pour l'accreditation du laboratoire. 34. Grodzielski M, Goette NP, Glembotsky AC, Constanza Baroni Pietto M, Mendez-Huergo SP, Pierdominici MS, et al. Multiple concomitant mechanisms contribute to low platelet count in patients with immune thrombocytopenia. Sci Rep. 2019 Feb 18;9(1):2208. PubMed PMID: 30778108. Pubmed Central PMCID: 6379541.

35. Rumjantseva V, Grewal PK, Wandall HH, Josefsson EC, Sorensen AL, Larson G, et al. Dual roles for hepatic lectin receptors in the clearance of chilled platelets. Nature medicine. 2009 Nov;15(ll): 1273-80. PubMed PMID: 19783995. Pubmed Central PMCID: PMC4428152.

36. Hoffmeister KM, Josefsson EC, Isaac NA, Clausen H, Hartwig JH, Stossel TP. Glycosylation restores survival of chilled blood platelets. Science. 2003 Sep 12;301(5639): 1531-4. PubMed PMID: 12970565.

37. Hezard N, Potron G, Schlegel N, Amory C, Leroux B, Nguyen P. Unexpected persistence of platelet hyporeactivity beyond the neonatal period: a flow cytometric study in neonates, infants and older children. Thrombosis and haemostasis. 2003 Jul;90(l): 116-23. PubMed PMID: 12876634.

38. Van Belle E, Rauch A, Vincent F, Robin E, Kibler M, Labreuche J, et al. Von Willebrand Factor Multimers during Transcatheter Aortic-Valve Replacement. The New England journal of medicine. 2016 Jul 28;375(4):335-44. PubMed PMID: 27464202.

39. Spillemaeker H, Dupont A, Kauskot A, Rauch A, Vincent F, Borgel D, et al. 5065 Platelet desialylation induced by high shear-stress mechanical circulatory support. European Heart Journal. 2018;39(suppl_l).

40. Arnold SV, Jones PG, Allen LA, Cohen DJ, Fendler TJ, Holtz JE, et al. Frequency of Poor Outcome (Death or Poor Quality of Life) After Left Ventricular Assist Device for Destination Therapy: Results From the INTERMACS Registry. Circulation Heart failure. 2016 Aug;9(8). PubMed PMID: 27507111. Pubmed Central PMCID: 4985017.

41. Jilaihawi H, Doctor N, Chakravarty T, Kashif M, Mirocha J, Cheng W, et al. Major thrombocytopenia after balloon-expandable transcatheter aortic valve replacement: prognostic implications and comparison to surgical aortic valve replacement. Catheterization and cardiovascular interventions : official journal of the Society for Cardiac Angiography & Interventions. 2015 Jan 1 ;85(1): 130-7. PubMed PMID: 24677617.

42. Van Belle E, Juthier F, Susen S, Vincentelli A, lung B, Dallongeville J, et al. Postprocedural aortic regurgitation in balloon-expandable and self-expandable transcatheter aortic valve replacement procedures: analysis of predictors and impact on long-term mortality: insights from the FRANCE2 Registry. Circulation. 2014 Apr 1;129(13): 1415-27. PubMed PMID: 24566199.

43. Dvir D, Genereux P, Barbash IM, Kodali S, Ben-Dor I, Williams M, et al. Acquired thrombocytopenia after transcatheter aortic valve replacement: clinical correlates and association with outcomes. Eur Heart J. 2014 Oct 07;35(38):2663-71. PubMed PMID: 24598983.

44. Sedaghat A, Falkenberg N, Sinning JM, Kulka H, Hammerstingl C, Nickenig G, et al. TAVI induces an elevation of hemostasis-related biomarkers, which is not causative for post-TAVI thrombocytopenia. International journal of cardiology. 2016 Oct 15;221:719-25. PubMed PMID : 27424316.

45. Yang Y, Peng X, Ying P, Tian J, Li J, Ke J, et al. AWESOME: a database of SNPs that affect protein post-translational modifications. Nucleic acids research. 2019 Jan 8;47(D1):D874-D80. PubMed PMID: 30215764. Pubmed Central PMCID: 6324025.

46. Giannini S, Lee-Sundlov MM, Rivadeneyra L, Di Buduo CA, Bums R, Lau JT, et al. beta4GALTl controls betal integrin function to govern thrombopoiesis and hematopoietic stem cell homeostasis. Nature communications. 2020 Jan 17;11(1):356. PubMed PMID: 31953383. Pubmed Central PMCID: 6968998.

Claims

CLAIMS:

1. A method for determining a reference range of b-galactose exposure platelet in a biological sample obtained from a subject comprising the steps of: i) determining the concentration of Ricinus communis agglutin (RCA) and the value of Forward SCatter (FSC) in said biological sample; and ii) calculating the ratio between RCA and FSC (RCA/FSC).

2. The method according to claim 1 further comprises a step of identifying non-specific populations appearing within the platelet population.

3. The method according to claims 1 to 2, wherein said biological sample is blood sample.

4. The method according to claim 1 to 4, wherein said biological sample is venous blood sample.

5. The method according to claims 1 to 4, wherein said subject is a healthy subject.

6. The method according to claims 1 to 5, wherein the RCA/FSC ratio comprises between 0.237 and 0.941.

7. The method according to claims 1 to 6 is suitable for measuring the number of platelet b-galactose exposure in a biological sample obtained from a subject.

8. The method according to claims 1 to 6 is suitable to determine the quality of platelets in a biological sample.

9. The method according to claims 1 to 6 is suitable to measure the quality of platelets during a transfusion.

10. A method for determining whether platelets in a biological sample obtained from a subject are desialylated comprising the steps of: i) calculating the ratio between RCA and FSC according to claim 1; and ii) concluding that platelets are desialylated or hyposialylated when the ratio is outside the range according to claim 6.

11. A method for diagnosing a platelet desialylation related disease in a subject comprising the steps of: i) calculating the ratio between RCA and FSC according to claim 1; and ii) concluding that the subject is suffering or is susceptible to suffer from a platelet desialylation related disease when the ratio is outside the reference range according to claim 6.

12. A method for predicting whether a subject suffering from a platelet desialylation related disease will achieve a response with a sialidase inhibitor comprising the steps: i) calculating the ratio between RCA and FSC according to claim 1; and ii) concluding that the subject will achieve a response with a sialidase inhibitor when the ratio is in the reference range according to the invention or concluding that the subject will not achieve a response with a sialidase inhibitor when the ratio is outside the reference range according to claim 6.

13. A method for treating a subject suffering from a platelet desialylation related disease, comprising the steps of: i) determining said subject will achieve a response with a sialidase inhibitor according to claim 11; and ii) administering said subject a sialidase inhibitor when said subject is determined as responder with a sialidase inhibitor treatment.

14. The method according to claims 12 to 13, wherein the platelet desialylation related disease is selected from the group consisting of but not limited to: thrombocytopenia, sepsis, immune thrombocytopenia (ITP), gestational thrombocytopenia, congenital disorders of glycosylation, VWF-type 2B disease, bacterial infections, viral infection (e.g. influenza virus A infection, dengue), parasitic infection (e.g. malaria), liver diseases (e.g. NAFLD, NASH), allogenic hematopoietic stem cell transplantation, disorders related to Trans Aortic Valve Implantation (TAVI) or Mechanical Circulatory Support Implantation (MCSI).

15. A kit for performing the method according to the invention, wherein said kit comprises i) means for identifying non-specific populations appearing within the platelet population (for example with a Mitotracker), ii) means for determining RCA and FSC in a biological sample obtained from a subject; and iii) instructions notice with a reference range.