The invention relates to the medical field in general and in particular the field of in vitro diagnostics. It relates more specifically to the extraction of analytes contained in a blood sample previously dried on blotting paper as used in the DBS technology.

There are now more than a century, Ivar Bang described a method of noninvasive capillary blood samples dried on blotting paper for estimating the glucose content as an alternative to conventional blood sampling, venous, stored in tubes. The technology, called "Dried Blood Spot" or DBS, is based on the deposition of a drop of blood on a suitable filter paper and dried before storage and analysis.

In 1963, Robert Guthrie et al. (A simpler method for Detecting phenylalanine phenylketonuria in broad populations of newborn infants Pediatrics.1963; 32. 338-343) used this technology to develop systematic neonatal screening for phenylketonuria. Setting up for the first time in Scotland, the use of DBS was extended in infants in the UK in the 70s, mainly to detect inborn errors of metabolism that can be a supported therapeutic. Currently, the use of DBS is almost exclusively limited to newborn screening.

In France, the capillary sampling and preservation DBS was established in 1978 by the French Association for screening and prevention of childhood disabilities. The collection of capillary blood from newborns also allows to detect phenylketonuria, detection of congenital hypothyroidism, congenital adrenal hyperplasia, cystic fibrosis or sickle cell anemia.

Thus, the principle of DBS technology is based on the collection of a blood sample, the application of the sample on blotting paper, conservation and extraction of analytes contained in the sample dried on blotting paper to perform analysis of these analytes.

At present, there is a capillary sampling is done at the finger or heel in infants. The skin is pierced using a tool such as a sterile lancet and drops of blood are deposited in printed circles on the blotting paper.

Once all printed circles are filled, the blotting paper was dried for several hours at room temperature.

Once dried, for conservation of the blotting papers are placed in a sealed plastic bag, optionally with a desiccant sachet and a moisture indicator. The desiccant is to complete the drying process to reduce the risk of blood sampling related infection. Possible storage time at room temperature depends on the nature of the analyte, one week for the proteins to a year or more for nucleic acids.

The extraction of analytes blotting papers must be performed using a standardized procedure. One or more blotter paper discs varying diameters are cut with a special cutter. These discs are then placed in a large volume of elution buffer for a variable period according to the extraction procedure. The nature and volume of the elution buffer plays a major role in the redissolution of the analytes to be tested. A wide variety of elution buffers has been described in the literature, the most common are saline buffers, often supplemented with detergent (Tween, triton), carrier proteins (albumin) and chelating agents (EDTA). The organic solvent (acetonitrile, methanol or ethanol) are also good elution buffers for

Thereafter, the DBS technology can be support for multiple analyzes. Indeed, from this technology analyzes such as serology, early pediatric molecular diagnostics, a quantification of the circulating viral load but also a resistance genotyping, can be realized.

Under a serodiagnosis, antibody detection can be carried out by ELISA or rapid tests after elution of DBS in buffer. This procedure is mainly used for epidemiological studies. This type of analysis requires small amounts of samples, about 50 μί.

The increase in biological diagnostic applications has encouraged the emergence of automata increasingly fast and reliable in the clinical laboratories. The immunoassay controllers allow the dosage of cardiac markers, thyroid, tumor, fertility, anemia, or the determination of infectious serology.

Using these immunoassay automation requires larger volumes of blood than is possible with a capillary blood sample. Indeed these automata

Immune analyzes the blood sample volume can range from a hundred to several hundred microliters for analysis.

Indeed, it should be noted that the blood samples collected to perform DBS are of the order of a few microliters. This is enough for some analyzes DNA in general, but is insufficient for analysis on immunoassay automation. The main drawback of DBS technology is related to low levels of blood collected available for testing, and the difficulties encountered in the laboratory to extract the maximum possible material without contamination (Dachraoui R et al., RNA amplification ofi the Pol and HIV-1 env region is dried serum and plasma spots, HIV Med 2008; 9:. 557-61).

At present, to extract maximum material from a DBS very high volumes of buffer are used. In the works of Dachraoui et al. or those of Borremans (Ammonium Improves elution fixed ofi dried blood spots without afifiecting immunofluorescence assay quality, Trop Med Int Health 2014 Apr; 19 (4):. 413-6) buffer volumes used are 10 to 30 times higher the sample volumes deposited and dried on the DBS. Thus recovered sample is found strongly diluted analytes relative to the original sample. This dilution of the original sample has the effect of reducing the recovered sample sensitivity analysis or even worse may alter the clinical status of the sample. Edit clinical status means that for an initial sample before drying on blotting paper, positive for a particular viral infection, the sample recovered after extraction as used in the DBS technology becomes negative for the same infection. Indeed an analyte present in small quantities in the deposited sample on the DBS may be under the detection threshold in a diluted sample.

Therefore to be analyzed on immunoassay automation using blotting paper as in DBS technology requires adaptations. First to retrieve sample volumes for the controller on analysis requires that the initial sample volume is sufficient. Thus it is necessary for it to work the paper to be able to absorb but also to restore a sufficient sample volume. All without the recovered sample is diluted with respect to the original sample.

A larger sample volume results in greater absorption and greater depth of the sample to be analyzed. The analytes are found therefore trapped between the fibers of blotting paper. Thus the main difficulty lies in extracting the analytes trapped in the filter paper, after the sample is dried, effectively without distorting these analytes to be analyzed thus keeping the clinical status of the original sample. Indeed it is crucial for an initial positive sample for viral infection, the sample recovered after extraction from DBS remains positive for the same infection. And in the same way if the original sample is negative for infection, the sample recovered after extraction should the

So in order to keep the clinical status, the process should allow to achieve both quantitative and qualitative extraction of analytes absorbed into the blotter.

In addition, it is important to note that the blood is a connective tissue, a complex medium consisting of liquid (plasma) cells (red cells, white cells and platelets), and fibers (the fibrinogen which has a role in the blood clotting). This constitution has an impact on the structure of analytes contained in the blood sample. Thus the clinical status also may be impacted by the constitution of the sample recovered after extraction. Indeed the sample recovered after extraction must have a construction similar to the original blood sample. That the recovered sample should mimic the blood matrix of the initial sample. The analytes of the environment in the extraction solution must be chemically close to their environment in the blood sample. This maintains the conformation space of these analytes. This is crucial in the context of an immunoassay that when the epitope is conformational said.

There is thus a real need for a method for efficient extraction and without denaturation of analytes contained in a blood sample dried on blotting paper for purposes of analysis automatons found in clinical laboratories while maintaining the clinical status of the original sample.

Thus, the present invention relates to a suspending process of the analytes contained in a blood sample previously dried on blotting paper, wherein:

on has a papier buvard sur me a model serum, a volume of initial upper or distinction to 100 μί, east Canary led papier buvard ayant une capacité d'absorption dudit model of au moins 60 μΐ - ,. αη 2 Clark and a grammage between 200 gm "2 and 800 gm " 2 ;

on transference led papier buvard in a récipient;

a liquid extraction solution is added in said container, said extraction solution being selected from:

o a saline phosphate buffer having a phosphate concentration of between 10 mmol.L "1 and 150 mmol.L " 1 and a sodium chloride concentration between 150 mmol.L "1 and 350 mmol.L " 1 and o tris buffer saline having a tris concentration of 10 mmol.L "1 and 150 mmol.L " 1 and a sodium chloride concentration between 150 mmol.L "1 and 350 mmol.L " 1 ,

said extraction solution having a volume equivalent to the volume of blood sample dried on blotting paper;

is allowed to impregnate said blotting paper in the extraction solution;

mechanical dewatering share is exerted on the paper contained in the container; and

recovering the extraction solution comprising the analyte contained in the original blood sample.

The applicant has therefore demonstrated, against all odds, that the method of the invention easy to implement and ergonomic, allows rendering a result maintaining clinical status (positive, negative or equivocal) of the sample.

Before going further in the description of the invention, the following definitions are provided to facilitate understanding.

In general, the term "sample" refers to a portion or quantity, especially a small part or a small amount, taken from one or more entities for analysis. This sample may optionally have been pretreated, for example involving the stages of mixing or dilution.

The sample under the method of the invention is a biological sample of human origin (or animal), corresponding to blood biological fluid samples (whole blood or derivatives such as serum or plasma).

The sample analyzed is generally susceptible to, or suspected, to contain at least one representative of analyte the presence of microorganisms or disease to detect, characterize or monitor.

The object of the present invention is the suspension of the analytes contained in a blood sample dried in a blotting paper according to the technology Dried Blood Spot (DBS).

The term analyte refers to a biological active substance, contained in a sample, detected, identified and / or quantified by analysis. It should be understood in the broad sense, as referring to a chemical, biological or biochemical which is the subject of one or more analyzes. Examples of analytes include a protein or peptide.

The analyte will be representative of a disease state. For medical condition is any condition altered a patient due to diseases caused by many factors such as environmental (infectious). Examples of diseases, infectious diseases include, caused by microorganisms such as influenza virus, dengue fever or Ebola. The analytes are then associated with various diseases. Examples of analyte include p24 antigen and anti-HIV-1 and HIV-2 antibodies useful as analytes for the AIDS virus or HBsAg and anti HBs as analytes of hepatitis B.

The analyte present in a sample is to be detected, quantified, analyzed, characterized, or evaluated.

Within the meaning of the present invention, the blotting paper is a porous paper capable of absorbing by capillarity a quantity of liquid.

Blotting paper, according to the present invention, means a fibrous material consisting partly or entirely of fibers. These fibers may be assembled in an orderly or randomly (that is to say a woven or non-woven). This material has the ability to absorb aqueous fluids may be composed of only one type of fiber, fiber mixture or may not belong to the same class.

The fibers may be classified according to their chemical composition (inorganic or organic) and their origin (natural or artificial). For example there may be mentioned glass fiber such as artificial mineral fiber or cellulosic fiber (cotton) as natural organic fibers of vegetable origin.

In the context of the invention the filter paper has a capacity of absorption of a blood sample of at least 60 μΙ ^ ΏΪ 2 . Is considered according to the invention that the blotter paper should be capable of absorbing a liquid composition with physical properties of viscosity of a sample of freshly drawn blood. For example the viscosity of the blood sample is between 4 to 25 x 10 -3 pascal-seconds (Pa.s).

According to the method of the invention the filter paper has a basis weight of 200 gm "2 and 800 gm " 2 . The weight, also called "strength of the paper," is a quantity

characterizing a paper or cardboard, corresponding to its mass per unit area, that is to say to its mass per unit area. The weight is defined by the ISO 536 and 4046 standards.

By "extraction solution" means a solution enabling the solubilization of the analyte from a blood sample dried in absorbent paper. This solution, as part of the invention has a dual function. Initially its role is to extract analytes absorbed into the blotter without denaturing them. Then this extraction solution by its very constitution, provides for mimic blood matrix in the ultimately intended to allow an analysis of PLCs. All this to keep the clinical status of the original sample.

According to the present invention the extracting solution is selected from phosphate-buffered saline (PBS) and tris buffer saline (TBS).

Phosphate buffered saline, or PBS, is a widely known buffer solution for the skilled person. This is a physiological saline containing sodium chloride, disodium phosphate, monopotassium phosphate and potassium chloride.

In the context of the present invention, the phosphate buffered saline has a phosphate concentration of between 10 mmol.L "1 and 150 mmol.L " 1 and a sodium chloride concentration between 150 mmol.L "1 to 350 mmol. The "1 .

The preparation of this type of solution is widely known in the art. For example, one can prepare a phosphate buffered saline solution of 1 liter having a phosphate concentration of lO mmol.L "1 and a sodium chloride concentration of 137 mmol.L " 1 pH 7.4 by dissolving:

1.44g of disodium hydrogen phosphate (Na 2 HP0 4 ) concentration of 10 mmol.L "1 ,

0.24g de phosphate de monopotassium (KH2P04) de concentration 2 mmol.L"1,

8g of sodium chloride (NaCl) concentration of 137 mmol.L "1 , and

0.2g potassium chloride (KC1) concentration 2.7 mmol.L "1 ,

in 800 ml of water (H 2 0), then adjust the pH to 7.4 with hydrogen chloride

(HC1) and fill with distilled water to 1 liter.

The saline tris, or TBS, is also a widely known buffer to the skilled person. This buffer contains trishydroxymethylaminomethane, or tris, and sodium chloride. In the context of the present invention the tris saline buffer has a

tris concentration between 10 mmol.L "1 and 150 mmol.L " 1 and a sodium chloride concentration between 150 mmol.L "1 and 350 mmol.L " 1 .

The preparation of this type of solution is widely known in the art. For example, one can prepare a tris salt buffer solution of 1 liter having a concentration of tris 50 mmol.L "1 and a sodium chloride concentration of 150 mmol.L " 1 pH 7.5 by dissolving:

6.05 g Tris concentration of 50 mmol.L "1 , and

8.76 g of sodium chloride (NaCl) concentration of 150 mmol.L "1 ,

in 800 ml of water (H 2 0), then adjusting the pH to 7.5 with hydrogen chloride (HC1) concentration of 1 mol L "1 and filling with distilled water up to 1 liter. in the context of the present invention, the volume of extraction solution is equivalent to the volume of original sample before drying in the blotting paper. this has the technical effect not to dilute the recovered sample using the method of the invention compared to the initial sample deposited and dried on blotting paper thereby to improve the sensitivity of the process and keep the sample clinical status.

Sensitivity is the percentage of "true positives" among all positive, recognized as such. It expresses the ability of the test to detect true positive biological samples, which correspond to pathology. In a "probabilistic" language, it corresponds to the probability of observing a positive result knowing the positive sample.

By "equivalent" is meant that the ratio between the lowest and the highest volume is between 0.8 and 1.

As previously explained the extraction solution in the context of the invention, two separate functions that act synergistically in order to permit storage of clinical status. The first is to enable the dissolution of analytes contained in a sample previously dried on blotting paper without denaturing them. And the second is to mimic the blood matrix to allow its analysis on immunoassay automation. The elements that make up this extraction solution enable it to fulfill these functions in the ultimately goal to maintain clinical status.

As illustrate in the Examples, the salt buffer main mission suspending analytes without denaturing them when sodium chloride at

concentrations between ISO mmol.L "1 and 350 mmol x L " 1 reproduces physiological conditions.

Advantageously and according to the invention the extraction solution has a pH between 7.2 and 7.5 and preferably between 7.3 and 7.4.

In a first embodiment the extraction solution further comprises one or more elements selected from the following list: a detergent, a carrier protein, an ion chelator and milk powder.

The detergents used in the laboratories of biology and biochemistry are mild surfactants used for the lysis of cell membranes and the dissolution of intracellular material in a soluble form. Their main applications are the dissociation of protein-protein interactions, protein-lipid and lipid-lipid, denaturation of protein structure and prevention of non-specific binding in immunochemical approaches and crystallization of proteins. Several types of detergents exist and are grouped according to their properties. The detergent promotes solubilization of analytes contained in a dried sample, trapped in the fibers of the filter paper. This dissolution must be done without altering the analytes.

According to a preferred embodiment of the invention, the detergent is a nonionic detergent and is selected from those usually used in the elution buffers and widely known to those skilled in the art. Examples include polyethylene glycol sorbitan monolaurate, also known as Tween 20 or Triton X-100.

The carrier protein's role is to recreate in the sample extract from the blotter the viscosity of a blood sample. Examples include the bovine serum albumin (BSA).

As for the reactive metal chelators such as EDTA or EGTA, they bind to Mg ions 2+ and thus prevent the cleavage of proteins by contaminating metalloproteinases. So their role is to allow to retain the conformation of analytes that will be included in the extraction solution. This has ultimately aim to keep the clinical status of the original sample.

In an alternative of the invention, the extraction solution comprises an organic solvent. The organic solvents used as elution buffer are widely known to those skilled in the art. Examples include acetonitrile, methanol or ethanol.

The term "spin" according to this invention is defined by the operation of removing a blotting paper a liquid that permeates through a mechanical action.

Finally the term "exert a dewatering effect on the blotting paper" refers to mechanical action or contact distance which can be localized or distributed can deform a body such as a fibrous material. In one embodiment of the invention, the duration of re-impregnation of the paper by the extraction solution is between 1 minute and 3 minutes, preferably is 2 minutes.

According to the invention embodiment, the blotting paper is a non-woven fibrous material comprising fibers selected from cellulose fibers and glass fibers, preferably of cellulose fibers.

Advantageously, the blotting paper has a thickness between 0.8 mm to

2,50 mm.

In one embodiment of the invention, the blotting paper is disc-shaped.

Preferably, the paper is dried at temperatures between 25 and 45 ° C and humidity percentages between 10 and 30%.

According to the invention the mechanical action is exerted on the blotting paper has a strength between 80 N and 120 N.

Advantageously, the mechanical wiping action on the paper by centrifugation, sonication or by automatic or manual mechanical pressure, preferably by manual mechanical pressure. The mechanical spin action is performed while maintaining the integrity of the paper. That this mechanical dewatering work is carried out without disintegrating paper.

According to one embodiment of the invention, the container is adapted to cooperate with a sliding means within said vessel, said means being impermeable to liquids and providing a seal with the side walls of the container.

Preferably, the blotting paper comprising dried blood sample is stored in a sealed container and the latter may contain a desiccant.

According to another embodiment of the invention, the method further comprises an analysis of the recovered sample comprising analytes by immunoassay.

Another object of the present invention is a method for analyzing a blood sample previously dried on blotting paper, said method comprising:

suspending of analytes contained in a blood sample previously dried on blotting paper implementing the suspending process of the analytes according to the invention, and

Analysis of the recovered extraction solution comprising the analyte contained in the original blood sample.

Analysis means determining the presence or absence of an analyte or determining the amount, or dose, of an analyte in the recovered extraction solution.

The determination of the presence or absence and the amount of an analyte can be as widely known techniques of the art. Examples include assays immunoassays such as ELISA (Enzyme Linked Immuno Sorbent Assay), ELFA (Enzyme Linked Fluorescent Assay) and RIA (radioimmunoassay).

The assay is an immunoassay method well known in the art and widely used in the field of the analysis of biological samples. It allows to detect analytes in samples such as proteins (antigen / antibody), peptides, and haptens, such as steroids or vitamins, involving logical immuno reactions between the analyte to be detected, and one or more partner (s) of binding to said analyte. In determining the amount, the immunoassay methods are based on measures to quantify the signals in the analysis of the biological sample. The amount of detected signals is generally proportional to the amount, or dose, of analyte to be measured (for example during

According to one embodiment the analysis of the sample to the extraction solution containing the analytes is performed by a technology selected from chemiluminescence, fluorescence, ELISA, EIA, by multiplexing, for biochips, immunochromatography, magnetic beads and radio -immunoanalyse.

Other objects, features and advantages of the invention will become apparent from the following description and examples discussed below, which refer to the accompanying figures and in which:

Figures 1 and 2 are schematic representations of a device 1 for exerting mechanical wiping action on the paper according to the method of the invention, and its principle of general use;

the Figures 3 is a schematic representation of another apparatus 2 also to take up the mechanical dewatering action and the general principle of use.

These examples are intended to facilitate understanding of the invention, its implementation and use. These examples are for illustrative purposes and should not limit the scope of the invention.

The invention will be better understood using the following examples are illustrative and not limiting.

EXAMPLES

I. Feasibility with the chickenpox virus

Chickenpox is a highly contagious childhood disease with a high prevalence in France (> 98%).

Illustration, sample analysis was performed using the automated immunoassay marketed by bioMérieux company, the VIDAS ® . The VIDAS ® VZG (varicella zoster) is an aid in the diagnosis of infection with varicella virus, it allows the detection of immunoglobulin G.

The VIDAS ® is a multi PLC immunoassays. It is a closed system for unit testing, offering great flexibility. This controller is characterized by its robustness, flexibility, ease of use and is designed for laboratories of small and medium size. It allows for routine testing, confirmation and tests with high medical value.

Detection is by the ELFA technique (Enzyme Linked Fluorescent Assay) in serum or plasma. The assay principle ELFA corresponds to the combination of reactions immunoenzymatic detection by endpoint fluorescence. The enzyme used is alkaline phosphatase, which catalyzes the hydrolysis reaction of the substrate, 4-methyl-ombelliferyl phosphate in a product; 4-methyl-ombellierone. The product emits at a wavelength of 450 nm after excitation at 370 nm. The results are automatically analyzed by the VIDAS ® and expressed as relative fluorescence intensity or RFV

(For "Relative Fluorescent Value"). This value of RFV is determined by subtracting the background (BKG) the gross value obtained.

Initially, it was studied the characteristics of the paper and the necessary extraction solution for the smooth application of the method according to the invention.

In this part, the goal will be the dissolution of the analytes of interest in the extraction solution for implementation in a VIDAS ® VZG.

To allow its installation in difficult conditions of collection, storage, transport and finally the analysis method according to the invention should be easy to implement, ergonomic and allow rendering a result maintaining the clinical status (positive, negative or equivocal) the sample.

a) paper absorption capacity

The paper 903 from Whatman ® is conventionally used when applying on blotting paper under the DBS technology, molecular biology. This thin paper is suitable for handling one to three drops of blood. In this case, a small amount is sufficient for the nucleic acids to look for are subjected to PCR amplification.

In a sero logical diagnostic context using an automated immunoassay it is necessary to work with a paper for depositing and recovering a greater volume of blood sample. The VIDAS ® , for example, require 100 serum test for the hepatitis C virus (HCV), 150 for the test on the hepatitis B virus (HBV) and 200 μΐ, for test the AIDS virus (HIV). These volumes are substantially equivalent to other tests on controllers on the market.

Thus, the paper must be able to absorb but also to restore a sufficient sample volume, immediately after drying or after a period of storage.

In order to select the most suitable paper to the method, a study was conducted on 6 paper types provided by the company Ahlstrom ® (TNF, 226, 222, 350, 270 and 320). These papers are characterized by their weight, their weight and thickness. TNF papers and 226 have similar characteristics to the paper from Whatman 903 ® which is conventionally used in the context of the DBS technology.

Reference (Grade) Composition Grammage (g / m 2 ) Thickness (mm)

167L 1 360 Cotton

Cotton 226 179 0.52

Cotton 222 291.3 0.82

Cotton 270 438.8 1.81

Cotton 320 702 2.47

Cotton 350 224.6 1.39

TNF Cotton 179 0.48

Table 1: Characteristics of paper thicknesses and schematic representation

The paper must have certain characteristics. Indeed, it must be flat surface and homogenous, allowing uniform distribution of the blood sample over its entire surface at the time of deposit.

In addition, the paper must be easily handled with reasonable estimated drying time to two hours, so do not be too thick.

The diameter of the discs is an important factor. Each paper has a volume limit is not exceeded because beyond the paper does not absorb the sample. It is therefore precisely define the diameter of the discs. The table below shows the maximum volume absorbable for paper grades 222 and 270.

The results show that variation of 0.5 mm on the diameter results in a decrease in the controlled absorption capacity. The volume deposited must permeate the entire paper but without overflow.

For further experiments, a paper disc-shaped 20 mm in diameter that deposits beyond 200 blood sample will be searched. Indeed handling larger diameter discs, 30 to 50 mm can quickly become difficult. Thus, the blotter paper according to the invention should have an absorption capacity of at least 60 μΊ, .αη 2 .

The various papers listed in Table 1 are tested with three volumes of different sera: 100, 200 and 500 μί. These volumes can meet the necessary test taken for a test on immunoassay automation.

Table 3: Circulation Diameter (mm) depending on the paper and the deposited volume

After deposition of the serum, the diffusion diameter for each paper was measured. Given the diameter selected for the paper, beyond 20mm diameter distribution of it is considered that the paper is no longer able to absorb the serum. In other words the paper should have an absorption capacity of at least 60 μί ι 2 of a blood sample.

The results show that the grades 270, 320, 350, 167L and 222 meet the requirements of the invention. While for the corresponding grades at 903 Whatman ® conventionally used in DBS, that is to say, the 226 and TNF, this is not the case.

b) Solution d'extraction

Then the extraction solution that enables recovery of analytes that have been absorbed into the fibers of the filter paper during drying was studied.

As indicated above, this solution has a dual function, namely to allow the extraction of analyte initially present in the starting sample and without denaturing them to mimic the blood matrix of the initial sample to allow analysis on automaton 'immunoassay. All this to keep the clinical status of the initial sample.

Thus, in the context of the invention a simple extraction solution formulation, the more physiological possible, do not degrade the entrapped analytes is desired.

Ten extraction solution formulations are tested. At first the impact of these solutions on a serum with evidence of their performance nontoxic diluent on the structure of analytes, is observed.

The study of non-toxicity of the extraction solution is performed by tests on pure serum and diluted half in each extraction solution. The non-toxicity of the solution is confirmed if the ratio between the pure serum RFV signal and that of the diluted half is less than two.

These extraction solutions were tested at different serum samples having different amounts of analytes. The table below shows the signal obtained on VIDAS ® for each pure serum sample and diluted in an extraction solution.

Serum 1 (> 500 RFV) 2 serum (> 500 RFV) serum 3 (> 1000 RFV) ratio (serum ratio (serum (serum

1 1/2 1 1/2 1 1/2

pure / serum diluted) / pure serum diluted) / pure diluted serum)

PBS50mMNaCl 154mM

758 495 1.53 695 401 1.73 1003 810 1.24 Tween200.05% BSA 5%

PBS50mMNaCl 154mM

758 555 1.37 695 515 1.35 1003 940 1.07 Triton 1% BSA 5%

PB S 50mMNaCl 154mM

758 476 1.59 695 396 1.76 1003 751 1.34% Urea 1MBSA5

PBS 50mM NaCl 308 mM

758 459 1.65 695 435 1.60 1003 784 1.28 Tween200.05% BSA 5%

PBS 50mMNaCl 154 mM

758 524 1.45 695 414 1.68 1003 799 1.26 Tween200.5% BSA 5%

PBS 50mMNaCl 154 mM

758 505 1.50 695 407 1.71 1003 805 1.25 Tween205% BSA 5%

PBS lOmMNaCl 154 mM

758 503 1.51 695 389 1.79 1003 779 1.29 Tween200.05% BSA 5%

PBS 150mMNaCl 154

mM Tween200.05% BSA 758 469 1,62 695 398 1,75 1003 853 1,18 5%

Tris lOmMNaCl 154 mM

758 421 1.80 695 358 1.94 1003 764 1.31 Régilait 0.3%

PBS 50mMNaCl 154 mM

758 401 1,89 695 377 1,84 1003 751 1,34 Tween200.05%

Serum 4 (> 1000 RFV)

ratio (serum

1 1/2

pure / diluted serum)

PBS50mMNaCl 154mM

1019 598 1,70

Tween200.05% BSA 5%

PBS50mMNaCl 154mM

1019 673 1,51

Triton 1% BSA 5%

PBS50mMNaCl 154mM

1019 621 1,64

Urea 1MBSA5%

PBS 50mM NaCl 308 mM

1019 590 1,73

Tween200.05% BSA 5%

PBS 50mM NaCl 154 mM

1019 647 1,57

Tween200.5% BSA 5%

PBS 50mM NaCl 154 mM

1019 671 1,52

Tween205% BSA 5%

PBS lOmMNaCl 154 mM

1019 625 1,63

Tween200.05% BSA 5%

PBS 150mMNaCl 154

mM Tween200.05% BSA 1019 573 1,78

5%

Tris lOmMNaCl 154 mM

1019 625 1,63

Régilait 0.3%

PBS 50mM NaCl 154 mM

1019 549 1,86

Tween200.05%

Table 4: Composition and efficiency of extraction buffer

In view of the above, the extraction solutions have no toxic effect on the sera. Thus these solutions do not alter the analytes in the sample while reproducing physiologic conditions allowing their analysis on automatic analyzers.

c) extraction devices

In order to solve the technical problem, namely the extract analytes absorbed in a filter paper but without denaturing them while maintaining the clinical status of the starting sample, the method of the invention comprises a mechanical action step d 'spinning.

To illustrate the invention and example only two devices are used to exert the mechanical action on the paper according to the method of the invention.

Both devices based on pressure extraction can recover sufficient volume to test VIDAS ® .

The first device shown in Figure 1, comprises a lever 1 and an element acting as a piston 2, impermeable to liquids and providing a seal with the side walls 3 of a container, adapted to cooperate with said container 3.

As shown in Figure 1, actuation of the lever 1 enables the piston office making member 2 to exert a mechanical wiping action on the blotting paper 3 impregnated with the extraction solution in the container 4. The extraction solution 5 after spinning action comprising the analytes contained in the original blood sample is collected in a container 6.

The second device, shown in Figures 2 and 3, comprises:

- a container 7 adapted to contain at least one blotting paper 8 and a liquid solution, said container having a flat bottom comprising a vent hole 9 located on its lower part for passing the extraction solution 14,

- an upper element 10 acting as a piston, impermeable to liquids and providing a seal with the side walls of the container 7, comprising a clamping lever 11 and provided with a thread 12 adapted to cooperate by screwing with the container 7, and

- a lower element 13 to recover the extraction solution 14 comprising the analytes contained in the original blood sample.

As shown in Figure 3A, the container 7 comprising the blotting paper 8 and the extraction solution 14, co-operates firstly with the upper member 10 by extraction office 14. Once these three elements together, the clamping lever 11 is lowered, as shown in FIG 3B, to allow the screwing of the upper member 10 with the container 7 and thus increasing the force exerted. 3C shows the recovered extraction solution 14 due to mechanical dewatering Action exerted on the blotting paper 8 by the upper element 10.

Both devices based on pressure extraction can recover sufficient volume greater than or equal to 100 μί, for VIDAS ® . The first system, which will be called device 1 thereafter is a vertical clamping conveyor for applying a uniform pressure of 100 N, allows to recover at least 70% of the volume of solution deposited, over 400 extracts 600 deposited. It allows reproducible extraction because it works regardless of the strength of the user. The second system, which will be called device 2 subsequently allows for him to recover at least 50% of the volume.

piston and on the other hand with the lower element 13 for recovering the solution

Table 5: Volume of extraction solution recovered depending on the device used

The device 1 and papers, with a diameter of 20 mm, grades 222, 350 and 270 will be used for the following experiment.

The serum sample of a HIV-positive patient with varicella, ie whose clinical status is positive for varicella, is tested initially on the VIDAS ® to determine the value of its signal. It will be called thereafter original signal. Secondly, there is provided according to the invention the step of depositing and drying on paper before implementing the step of extraction using the extraction solution, the volume of which is equivalent to the volume of deposited sample, and the device 1, then the VIDAS ® . The result gave the VIDAS ® will pick up a signal. The value of the original signal and of those recovered in RFV signals,

Table 6: Signal Recovery Testing and clinical status of conservation

The results show that the extracted sample, using the extraction solution and the device 1, is sufficient to allow the VIDAS ® VZG. Without dilution of the sample, that is to say that the initial sample volume is exactly the same as the volume of extraction solution (settled volume = volume of extraction solution), the recovered signal remains important . Indeed it remains above 80% of the original signal.

Moreover, the clinical status of the recovered sample is the same as that of the initial sample, that is to say positive. That sample recovered after the extraction step remains HIV chickenpox as the original sample. Thus the clinical status is well preserved.

These results also show that two minutes of the paper re-impregnation time for the extraction solution are sufficient to obtain a satisfactory signal (greater than 80%>). This time allows the extraction solution to diffuse into the paper fibers which is rehydrated and better, which improves the release of trapped analytes.

The various tests carried out in parallel with other extraction solutions, listed in Table 4, show that they also allow to obtain a higher signal to 80% of the original signal (not shown).

Therefore the method of the invention retains the clinical status of an initial sample in connection with the use of DBS technology PLC

immunoassay. Indeed, despite the higher volumes placed on blotting paper (300, 350 and 500 μί), 80% of the analytes initially contained in the starting sample are present in the samples recovered what keeps the clinical status of the initial sample .

In order to demonstrate the reproducibility of the method according to the invention, the inventors have shown that it also works under other detection types of infections.

For the following tests, the same experimental conditions as those shown in Table 6 are used.

II. Transposition of the method according to the invention on HIV test

In order to demonstrate the reproducibility of the process, the diagnosis of the AIDS virus is studied.

The HIV (HIV-1 and HIV-2) are retro RNA viruses. This is an infection of the CD4 + (T-cells, macrophages).

Diagnosis of infection with this virus is based on the simultaneous detection of HIV p24 antigen-1, present the first three weeks between the time of infection and the onset of antibodies and detection of anti HIV- 1 and HIV-2, which allows monitoring of the seroconversion window.

Given the above results, the method of the invention allows the diagnosis for net positive samples. Indeed, with a process for the recovery of 60-70% sample extract and 80%> RFV response, the risk of a change in clinical interpretation, negative positive status is unlikely. The situation is different for weak and equivocal positive samples which are more easily subject to change clinical status, which is the case in connection with a diagnosis of AIDS.

Illustration, analyzes samples using the automated immunoassay marketed by bioMérieux company, the VIDAS ® , are performed. The kit VIDAS ® HIV DUO Quick, which allows the diagnosis of the virus requires

high test portion (200 μί) and is suitable for detecting very small quantities of analytes.

We want to be able to track the infection closest to contamination, so when the antibody level is still low. For the kit VIDAS ® HIV DUO Quick, the index was off limits {) corresponding to the positivity threshold is 125 VPN. In other words a sample with an index above 125 VPN is diagnosed as HIV positive to the AIDS virus.

For this experiment, 500 μΐ, sera are deposited on paper, diameter 20 mm and of 222 degree, and dried for hour 30 minutes at 30 ° C. For the extraction step, is allowed to impregnate the paper in 500 of extraction solution (PBS 50 mM NaCl 154 mM Tween 20 0.05%) for a period of 2 minutes. Both devices are used for mechanical dewatering step Action on paper.

The serum sample used for the experiment is seropositive for HIV. The value of the initial signal and those of VPN recovered signals, clinical status and signal percentage are presented in the table below.

The results show that the recovered signal is around 70%> after use of the method according to the invention. The recovered signal is lower than that obtained in VIDAS ® VZG nevertheless it still keeps the original clinical status. Indeed, samples recovered following the extraction step remains HIV positive.

III. Transposition of the method according to the invention on HBV and HCV test

The Hepatitis B Virus (HBV) is a DNA virus composed of an outer lipid envelope and a nucleocapsid protein containing the viral DNA. The envelope contains HBs proteins which are anchored and exposed at the virus surface in order to allow the steps of attachment and penetration into hepatocytes.

Following HBV infection, HBsAg (HBsAg) appears after several days and may persist for several months (= chronic infection). Usually the disappearance of HBsAg is followed by the appearance of anti-HBs. The research antibodies is performed in follow-infected patients but also to check the efficacy of HBV vaccination.

The sample analyzes were performed using the automated immunoassay marketed by the company bioMérieux VIDAS system ® and VIDAS ® HBs Ag.

For detection of HBsAg of hepatitis B in the kit VIDAS ® HBs Ag, 150 serum are needed. The test used is the kit VIDAS ® HBs Ag Ultra sold by the company bioMérieux, which defines a positive threshold value to 80 RFV. The threshold is very low in the reading scale VPN VIDAS ® .

500 sera are deposited on paper, diameter 20 mm and of 222 degree, and dried for hour 30 minutes at 30 ° C. The paper impregnating step is performed in 500 of extraction solution (PBS 50 mM NaCl 154 mM Tween 20 0.05%) for a period of 2 minutes. The devices 1 and 2 are used for mechanical dewatering step Action on paper.

The serum sample used for the experiment is HIV positive for the hepatitis B The value of the initial signal and those recovered in RFV signals, clinical status and signal percentage are presented in the table below.

Table 8 Study of the detection limit for HBV

The results show that a serum sample characterized as being close to the positivity threshold, clinical status remains positive for HBV in all scenarios.

IV. Transposition of the method of the invention on the HCV test

The hepatitis C virus (HCV) discovered by molecular biology methods, affects all countries. The HCV infection often develops in chronic hepatitis C (80%), exposing the infected person to the risk of liver complications. The kit VIDAS ® Anti-HCV, marketed by bioMérieux, is a qualitative test for the detection of immunoglobulin G antibodies against this virus. For this test the index of the positive threshold is very high, it is set to 1745 RFV.

For this experiment, 500 sera are deposited on paper, diameter 20 mm and of 222 degree, and dried for hour 30 minutes at 30 ° C. For the extraction step, is allowed to impregnate the paper in 500 of extraction solution (PBS 50 mM NaCl 154 mM Tween 20 0.05%>) for a period of 2 minutes. Both devices are used for mechanical dewatering step Action on paper.

The serum sample used for the experiment is seropositive for the virus of hepatitis C. The value of the initial signal and those recovered in RFV signals, clinical status and signal percentage are presented in the table below.

Table 9: Study of detection limit for HCV

In this case the limit index is very high (1745 RFV), however we can notice that the status remains the same between the initial sample and the sample recovered following the method of the invention. The latter is applicable.

CLAIMS

1. A method of suspending the analytes contained in a blood sample previously dried on blotting paper, wherein:

on has a papier buvard sur me a model serum, a volume of initial upper or distinction to 100 μί, east Canary led papier buvard ayant une capacité d'absorption dudit model of au moins 60 μΕ ι "2 and a compris entre grammage 200 gm "2 and 800 gm " 2 ;

on transference led papier buvard in a récipient,

a liquid extraction solution is added in said container, said extraction solution being selected from:

o a saline phosphate buffer having a phosphate concentration of between 10 mmol.L "1 and 150 mmol.L " 1 and a sodium chloride concentration between 150 mmol.L "1 and 350 mmol.L " 1 and o tris buffer saline having a tris concentration of 10 mmol.L "1 and 150 mmol.L " 1 and a sodium chloride concentration between 150 mmol.L "1 and 350 mmol.L " 1 ,

said extraction solution having a volume equivalent to the volume of blood sample dried on blotting paper;

is allowed to impregnate said blotting paper in the extraction solution,

mechanical dewatering share is exerted on said blotting paper; and

recovering the extraction solution comprising the analyte contained in the original blood sample.

2. The method of claim 1, wherein the pH of the extraction solution is between 7.2 and 7.5, preferably between 7.3 and 7.4.

3. The method of claim 1 or 2, wherein the extraction solution further comprises a detergent and / or carrier protein and / or chelating ion and / or milk powder.

4. A method according to any one of claims 1 to 3, wherein the step of impregnating the paper into the extraction solution has a duration between 1 minute and 3 minutes, is preferably 2 minutes.

5. A method according to any one of claims 1 to 4, wherein the blotting paper is a non-woven fibrous material comprising fibers selected from cellulose fibers and glass fibers.

6. A method according to any one of claims 1 to 5, wherein the blotting paper has a thickness between 0.8 mm to 2.50 mm.

7. A method according to any one of claims 1 to 6, wherein the blotting paper is disc-shaped.

8. A method according to any one of claims 1 to 7, wherein the mechanical wiping action on the blotting paper is made by manual mechanical pressure.

9. A method according to any one of claims 1 to 8, wherein the container is adapted to cooperate with a sliding means within said vessel, said means being impermeable to liquids and providing a seal with the side walls of the container.