OA19140A

OA19140A - Method for depositing a blood sample on a blotting paper and the subsequent mechanical extraction of the blood sample.

Info

Publication number: OA19140A
Application number: OA1201900231
Authority: OA
Inventors: Frédéric FOUCAULT; Ghislaine BOUCHARD-LOVIGHI
Original assignee: bioMérieux
Priority date: 2016-12-19
Filing date: 2017-12-19
Publication date: 2020-01-31

Abstract

The present invention relates to the medical field in general, and in particular the field of in vitro diagnosis. It relates more specifically to the extraction of analytes contained in a blood sample previously dried on blotting paper as used in the DBS technique. In this context, it proposes a method for suspending the analytes contained in a blood sample previously dried on blotting paper, wherein : blotting paper is provided on which a blood sample, with an initial volume of greater than or equal to 100 μl, is dried, said blotting paper having a capacity for absorption of said sample of at least 60 μl.cm-2 and a basis weight of between 200 g.m-2 and 800 g.m-2 ; said blotting paper is transferred into a container ; a liquid extraction solution is added to said container, said extraction solution being chosen from: a phosphate buffered saline having a phosphate concentration of between 10 mmol.l-1 and 150 mmol.l-1 and a sodium chloride concentration of between 150 mmol.l-1 and 350 mmol.l-1 and a tris-buffered saline having a tris concentration of 10 mmol.l-1 and 150 mmol.l-1 and a sodium chloride concentration 150 mmol.l-1 and 350 mmol.l-1 , said extraction solution having a volume equivalent to the volume of blood sample dried on the blotting paper; said blotting paper is left to impregnate in the extraction solution ; a mechanical draining action is applied to said blotting paper; and the extraction solution comprising the analytes contained in the initial blood sample is recovered.

Description

METHOD FOR SUSPENDING ANALYTES CONTAINED IN A BLOOD SAMPLE

PREVIOUSLY DRIED ON BLOTTING PAPER

The présent invention 'e laies to the medical field in general, and in particular the field of in vitro diagnosis. It r dates more specifically to the extraction of the analytes contained in a blood sample previously dried on blotting paper as used in the DBS technique.

More than a century ago now, Ivar Bang described a method for taking a noninvasive capillary blood sample dried on blotting paper for estimating the glucose content as an alternative to the conventional Dried Blood Spot or DBS, is t, paper which is dried before storage and analysis.

In 1963, Robert Guth phenylketonuria in large popida this technique to develop System the first time in Scotland, the us the 1970s, mainly for detecting innate metabolism errors which can be subjected to therapeutic treatment. Currently.

screening.

In France, the capillary blood sample and the préservation thereof on blotting paper was implemented in 1978 by tic Association française de dépistage et de prévention des handicaps de l’enfant [French Association for screening and preventing child disabilities], capillary blood sample from newborns allows, in addition to the détection of ia, the détection cf congénital hypothyroidism, congénital adrenal hyperplasia, venous blood sample stored in tubes. This technique, called ased on the deposit of a drip of blood on a suitable blotting rie et al. (A simple phenylalanine method for detecting ions of newborn infants. Pediatrics.1963; 32: 338-343) used itic néonatal screening for phenylketonuria. Implemented for iî of DBS was extended to infants in the United Kingdom in use of DBS remains almost exclusively limited to néonatal

Taking a phenylketonuria, cystic fibrosis or else sickle cell disease.

Thus, the principle of the DBS technique is based on the taking of a blood sample, the application ofthe blood san extraction ofthe analytes contained in this sample dried on blotting paper m order to perform pie taken to a blotting paper, the storage thereof and then the an analysis of these analytes.

At the current time, t the finger or and then drops of blood are printed circles are filled, the blc he capillary sample is a capillary sample which is taken from from the heel in i liants. The skin is pierced with a tool such as a stérile lancet dcposited in circles printed on the blotting paper. Once ail the tting paper is dried for a few hours at ambient température.

Once dried, in order to be stored, the blotting papers are placed in a sealed plastic bag, optionally with a sachet of desiccant and a moisture indicator. The aim of the desiccant is to finish the drying process in order to reduce any risk of infection linked to the blood sample. The possible storage time at ambient température varies according to the nature of the analyte, from one week for proteins to one year or more for nucleic acids.

The extraction of the analytes from the blotting papers must be carried out using a standardized procedure. One or more disks of blotting paper of variable diameters are eut out with a spécifie hole punch. These disks are then placed in a large volume of elution buffer for a variable period of time depending on the extraction procedure. The nature and the volume of the elution buffer plays a major rôle in the redissolving of the analytes to be tested. A large variety of elution buffers hâve been described in the literature, the most common of which are saline buffers, often supplemented with détergents (tween, triton), carrier proteins (albumin) and chelating agents (EDTA). Organic solvents (acetonitrile, methanol or éthanol) are also good elution buffers for extraction of proteins for example.

Subsequently, the DBS technique can be the basis for several analyses. Indeed, using this technique, analyses such as serodiagnosis, early pédiatrie molecular diagnosis, circulating viral load quantification, but also résistance genotyping, can be carried out.

In the context of serodiagnosis, the antibody détection can be carried out by ELISA or rapid tests after elution of the DBSs in a buffer. This procedure is especially used for epidemiological studies. This type of analysis requires small amounts of samples, of about 50 μΐ.

The increase in demands for biological diagnostics has promoted the emergence of increasingly fast and reliable automated devices in medical testing laboratories. Automated immunoanalysis devices make it possible to assay cardiac, thyroid, tumor, fertility or anémia markers or else to détermine infectious sérologies.

The use of these automated immunoanalysis devices requires larger volumes of blood than are allowed by a capillary blood sample. Indeed, in these automated immunoanalysis devices, the blood sample volumes can range from about a hundred up to several hundreds of microliters for one analysis.

Indeed, it is important to note that the blood samples collected for realizing a DBS are of a few microliters. This amount is sufficient for some analyses on DNA in general, but proves to be insufficient for analysis on automated immunoanalysis devices. The main drawback of the DBS technique is linked to the small amounts of collected blood available for the tests, and to the difficulty encountered in laboratories in extracting the maximum possible material therefrom without risk of contamination (Dachraoui R et al., RNA amplification of the HIV-1 Pol and env régions on dried sérum andplasma spots, HIV MED., 2008; 9:55761).

At the current time, to extract a maximum amount of material from a DBS, very high buffer volumes are used. In the studies by Dachraoui et al., or else those by Borremans {Ammonium improves elution of fixed dried blood spots without affecting immunofluorescence assay quality, Trop Med Int Health. 2014 Apr; 19(4):413-6), the buffer volumes used are 10 to 30 times higher than the volumes of sample deposited and dried on the DBS. Thus, the sample recovered is greatly diluted in terms of analytes compared with the initial sample. The conséquence of this dilution of the initial sample is a decrease in the sensitivity ofthe analysis of the recovered sample or, even worse, a risk of modifying the clinical status of the sample. Modifying the clinical status means that, for an initial sample which, before drying on blotting paper, is positive for a given viral infection, the sample recovered after an extraction as used in the DBS technique becomes négative for this same infection. Indeed, an analyte présent in low amount in the sample deposited on the DBS could be under the détection threshold in a diluted sample.

Consequently, in order to be analyzed on automated immunoanalysis devices, the use of blotting paper as in the DBS technique requires adaptations. First of ail, in order to recover large volumes of sample for the analysis on an automated device, the initial volume of sample must be sufficient. Thus, it is necessary, in order for that to work, for it to be possible for the paper to be capable of absorbing but also retuming a sufficient volume of sample. This must ail be done without the sample recovered being diluted compared with the initial sample.

A larger volume of sample results in a greater and deeper absorption of the sample to be analyzed. The analytes are consequently trapped between the fibers of the blotting paper. Thus, the main difficulty lies in extracting these analytes trapped in the blotting paper, after drying the sample, efficiently without denaturing these analytes to be analyzed, thus making it possible to preserve the clinical status of the initial sample. Indeed, it is essential that, for an initial sample that is positive for a viral infection, the sample recovered after extraction from a DBS remains positive for this same infection. And likewise, if the initial sample is négative for an infection, the sample recovered after extraction must also be négative.

Thus, in order to be able to preserve the clinical status, the method must make it possible to carry out an extraction, which is both quantitative and qualitative, of the analytes absorbed into the blotting paper.

Furthermore, it is important also to note that blood is a connective tissue, a complex medium consisting of liquid (plasma), cells (red blood cells, white blood cells and platelets) and fibers (fibrinogen which has a rôle in blood coagulation). This constitution has an impact on the structure of the analytes contained in the blood sample. Thus, the clinical status also risks being impacted by the constitution of the sample recovered after extraction. Indeed, the sample recovered after extraction must hâve a constitution similar to the initial blood sample. In other words, the sample recovered must mimic the blood matrix of the initial sample. The environment of the analytes in the extraction solution must be chemically close to their environment in the blood sample. This makes it possible to preserve the space conformation of these analytes. This is of essential importance in the context of an immunoassay when the epitope is said to be conformationaL

There is thus a real need to hâve a method which allows efficient extraction, without dénaturation, of the analytes contained in a blood sample dried on blotting paper for the purposes of analyses on automated devices présent in medical testing laboratories, while at the same time preserving the clinical status of the initial sample.

Thus, a subject of the présent invention is a method for suspending the analytes contained in a blood sample previously dried on blotting paper, wherein:

blotting paper is provided on which a blood sample, with an initial volume of greater than or equal to 100 μΐ, is dried, said blotting paper having a capacity for absorption of said sample of at least 60 pl.cm’² and a basis weight of between 200 g.m’² and 800 g.m’²;

- said blotting paper is transferred into a container;

a liquid extraction solution is added to said container, said extraction solution of being chosen from:

o a phosphate-buffered saline having a phosphate concentration of between 10 mmol.l’¹ and ISOmmol.l’¹ and a sodium chloride concentration of between 150 mmol.l’¹ and 350 mmol.l’¹ and o a Tris-buffered saline having a Tris concentration of between 10 mmol.l’¹and 150 mmol.l’¹ and a sodium chloride concentration of between 150 mmol.l’¹ and 350 mmol.l’¹, said extraction solution having a volume équivalent to the volume of blood sample dried on the blotting paper;

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

- a mechanical draining action is applied to the paper contained in the container; and

- the extraction solution comprising the analytes contained in the initial blood sample is recovered.

The applicant has thus demonstrated, against ail expectations, that the method according to the invention, which is easy to carry out and ergonomie, makes it possible to render a resuit preserving the clinical status (positive, négative or equivocal) of the sample.

Before going further with the description of the invention, the définitions below are given in order to facilitate the understanding thereof.

In general, the term sample refers to a portion or to an amount, more particularly a small portion or a small amount, taken from one or more entities for the purposes of analysis. This sample may optionally hâve undergone a prior treatment, involving for example mixing or diluting steps.

The sample in the context of the method of the invention is a biological sample, of human (or animal) origin, corresponding to samples taken of blood biological fluid (whole blood or dérivatives such as sérum or plasma).

The sample analyzed is, in general, capable or suspected, of containing at least one analyte représentative of the presence of microorganisms or of a disease to be detected, characterized or monitored.

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

The term analyte dénotés a substance of biological origin, contained in a sample, detected, identified and/or quantified by an analysis. In the broad sense, it should be understood as denoting a chemical, biological or biochemical substance which is a subject of one or more analyses. By way of example of analytes, mention may be made of a protein or a peptide.

The analyte will be représentative of a pathological condition. The term pathological condition is intended to mean any impaired state of health of a patient, due to diseases caused by numerous factors, such as environmental (infectious) factors. By way of example of diseases, mention may be made of infectious diseases, due to microorganisms such as influenza virus, dengue virus or else Ebola virus. The analytes are therefore associated with various diseases. By way of example of an analyte, mention may be made of the p24 antigen and anti-HIV-l and -HIV-2 antibodies that are of use as analytes for the AIDS virus, or else the HBs antigen and the anti-HBs antibody as analytes of hepatitis B virus.

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

For the purpose of the présent invention, the blotting paper is a porous paper capable of absorbing a certain amount of liquid by capillary action.

A blotting paper, according to the présent invention, is understood to be a fibrous material partially or totally consisting of fibers. These fibers can be assembled in an ordered or random manner (i.e. a woven or a nonwoven). This material which has the capacity to absorb aqueous liquids may be composed of a single type of fiber, of a mixture of fibers belonging to the same class or of a mixture of fibers not belonging to the same class.

The fibers may be categorized according to their chemical composition (minerai or organic) and their origin (natural or artificial). By way of example, mention may be made of glass fiber as artificial minerai fiber or else cellulose-based fiber (cotton) as natural organic fiber of plant origin.

In the context ofthe invention, the blotting paper has a capacity for absorption of a blood sample of at least 60 pl.cm'². It is considered, according to the invention, that the blotting paper must be capable of absorbing a liquid composition having the physical properties of viscosity of a freshly taken blood sample. For example, the viscosity of the blood sample is between 4 to 25 x HT³ pascal-seconds (Pa.s).

According to the method of the invention, the blotting paper has a basis weight of between 200 g.m’² and 800 g.m^-2. The basis weight, also called force of the paper, is a quantity characterizing a paper or cardboard, corresponding to its weight per unit area, that is to say its weight per unit of surface area. The basis weight is defined by the standards ISO 536 and 4046.

The term extraction solution is intended to mean a solution which makes it possible to dissolve the analytes from a blood sample dried in blotting paper. This solution, m the context of the invention, has a dual function. Firstly, it has the rôle of extracting the analytes absorbed into the blotting paper without denaturing them. This extraction solution then, by virtue of its constitution, must make it possible to mimic the blood matrix for the purposes. in the end, of allowing an analysis thereof on automated devices, ail of this making it possible to preserve the clinical status ofthe initial sample.

According to the présent invention, the extraction solution is chosen from phosphate-buffered saline (PBS) and Tris-buffered saline (TBS).

Phosphate-buffered saline, or PBS, is a buffer solution widely known to those skilled in the art. It is a physiological soluté containing sodium chloride, disodium phosphate, monopotassium phosphate and potassium chloride.

In the context of the présent invention, the phosphate-buffered saline has a phosphate concentration of between 10 mmol.l'¹ and 150 mmol.T¹ and a sodium chloride concentration between 150 mmol.1¹ and 350 mmol.l¹.

The préparation of this type of solution is widely known to those skilled in the art. By way of example, it is possible to préparé a phosphate-buffered saline solution of 1 liter having a phosphate concentration of 10 mmol.l'¹ and a sodium chloride concentration of 137 mmol.l'¹ with a pH of 7.4, by dissolving:

- 1.44 g of disodium phosphate (Na2HPÛ4) having a concentration of 10 mmol.l¹,

0.24 g of monopotassium phosphate (KH2PO4) having a concentration of 2 mmol.l’¹,

- 8 g of sodium chloride (NaCl) having a concentration of 137 mmol.l¹, and

- 0.2 g of potassium chloride (KC1) having a concentration of 2.7 mmol.l'¹, in 800 ml of water (H₂O), then to adjust the pH to 7.4 using hydrogen chloride (HCl) and make the volume up to 1 liter with distilled water.

Tris-buffered saline, or TBS, is also a buffer solution widely known to those skilled in the art. This buffer contains trishydroxymethylaminomethane, or Tris, and sodium chloride. In the context of the présent invention, the Tris-buffered saline has a Tris concentration of between 10 mmol.l'¹ and 150 mmol.l'¹ and a sodium chloride concentration of between 150 mmol.1'¹ and 350 mmol.l'¹.

The préparation of this type of solution is widely known to those skilled in the art. By way of example, it is possible to préparé a Tris-buffered saline solution of 1 liter having a Tris concentration of 50 mmol.l'¹ and a sodium chloride concentration of 150 mmol.l'¹ with a pH of 7.5, by dissolving:

- 6.05 g of Tris having a concentration of 50 mmol.l¹, and

- 8.76 g of sodium chloride (NaCl) having a concentration of 150 mmol.l'¹, in 800 ml of water (H2O), then by adjusting the pH to 7.5 using hydrogen chloride (HCl) having a concentration of l moLl'¹ and making the volume up to 1 liter with distilled water.

In the context of the présent invention, the volume of extraction solution is équivalent to the volume of initial sample before drying in the blotting paper. This has the technical effect of not diluting the sample recovered by means of the method according to the invention compared with the initial sample deposited and dried on the blotting paper, thereby making it possible to improve the sensitivity of the method and to preserve the clinical status of the sample.

The sensitivity represents the percentage of true positives among ail ofthe positives, acknowledged as such. It is expresses the ability of the test to detect the biological samples which are actually positive, and which correspond to the pathological condition. In probabilistic language, it corresponds to the probability of observing a positive resuit knowing the sample to be positive.

The term équivalent is intended to mean that the ratio between the lowest volume and the highest volume is between 0.8 and 1.

As previously explained, the extraction solution, in the context ofthe invention, has two distinct functions which act synergistically with the aim of enabling the clinical status to be preserved; the fïrst being to enable the dissolution of the analytes contained in a sample previously dried on blotting paper without denaturing them, and the second being to mimic the blood matrix so as to allow its analysis on automated immunoanalysis devices. The éléments which constitute this extraction solution allow it to perform these two functions for the purpose, ultimately, of preserving the clinical status.

As illustrated in the examples, the main mission of the saline buffer is the suspending of the analytes without denaturing them when the sodium chloride at concentrations of between 150 mmol.l’¹ and 350 mmol.l¹ makes it possible to reproduce physiological conditions.

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

In a fïrst embodiment, the extraction solution also comprises one or more éléments chosen from the following list: a detergent, a carrier protein, an ion-chelating agent and milk powder.

The détergents used in biology and biochemistry laboratories are mild surfactants used for the lysis of cell membranes and the dissolving of the intracellular material into a soluble form. Their main applications are the dissociation of the protein-protein, protein-lipid and lipid-lipid interactions, the dénaturation of the protein structure and the prévention of nonspecific bonds in immunochemical approaches and protein crystallization approaches. There are several types of détergents and they are grouped together according to their properties. The detergent promotes the dissolving of the analytes, contained in a dried sample, that are trapped in the fibers of the blotting paper. This dissolution must be carried out without denaturing the analytes.

According to one preferential embodiment of the invention, the detergent is a nonionic detergent and is chosen from those normally used in elution buffers and widely known to those skilled in the art. By way of example, mention may be made of polyethylene glycol sorbitan monolaurate, also known as Tween 20, or else Triton X-100.

The rôle of the carrier protein is to recreate, in the sample extracted from the blotting paper, the viscosity of a blood sample. By way of example, mention may be made of bovine sérum album in (BSA).

As regards the metal-chelating reagents such as EDTA or EGTA, they bind to Mg²⁺ions and thus prevent the cleavage of proteins by contaminating métalloprotéases. Their rôle is thus to make it possible to preserve the conformation of the analytes that will be taken up in the extraction solution. This has the objective, ultimately, of preserving the clinical status of the initial sample.

In one 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. By way of example, mention may be made of acetonitrile, le methanol or else éthanol.

According to the présent invention, the term draining is intended to mean the operation consisting in removing from a blotting paper a liquid with which it is impregnated, by means of a mechanical action.

Finally, the expression applying a draining action to the blotting paper refers to a remote mechanical action or a contact mechanical action which may be localized or distributed and which is capable of deforming a body such as a fibrous material. In one embodiment of the invention, the duration of re-impregnation of the paper with the extraction solution is between l minute and 3 minutes, preferably is 2 minutes.

ΙΟ

According to one implémentation variant of the invention, the blotting paper is a nonwoven fibrous material comprising fibers chosen from celluloses fibers and glass fibers, preferentially celluloses fibers.

Advantageously, the blotting paper has a thickness of between 0.8 mm and

2.50 mm.

In one embodiment of the invention, the blotting paper is in the form of a disk.

Preferably, the paper is dried at températures of between 25 and 45°C and at moisture percentages of between 10 and 30%.

According to the invention, the mechanical action is applied to the blotting paper at a force of between 80 N and 120 N.

Advantageously, the mechanical draining action on the paper is carried out by centrifugation, by sonication or by automatic or manual mechanical pressure, preferably by manual mechanical pressure. The mechanical draining action is carried out while at the same time preserving the integrity of the paper. ïn other words, said mechanical draining action is carried out without the paper disintegrating.

According to one embodiment of the invention, the container is capable of cooperating with a means sliding inside said container, said means being imperméable to liquids and providing a seal with the side walls ofthe container.

Preferentially, the blotting paper comprising the dried blood sample is stored in a sealed réceptacle and the latter may contain a moisture absorber.

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

Another subject ofthe présent invention is a method for analyzing a blood sample previously dried on blotting paper, said method comprising:

suspending the analytes contained in a blood sample previously dried on blotting paper by carrying out the method for suspending analytes according to the invention, and analyzing the recovered extraction solution comprising the analytes contained in the initial blood sample.

The terni analysis is intended to mean the détermination of the presence or absence of an analyte or the détermination of the amount, or dose, of an analyte in the recovered extraction solution.

H

The détermination of the presence or absence and of the dose of an analyte can be carried out according to the techniques widely known to those skilled in the art. By way of example, mention may be made of assaying by immunoassays, such as ELISA (Enzyme Linked Immuno Sorbent Assay), ELFA (Enzyme Linked Fluorescent Assay) and RIA (Radio Immuno Assay).

Assaying by immunoassay is a method well known to those skilled in the art and widely used in the field of biological sample analysis. It makes it possible to detect analytes in samples in the form in particular of proteins (antigens/antibodies), of peptides and of haptens, for instance steroids or vitamins, involving immunological reactions between the analyte to be detected and one or more partner(s) for binding to this analyte. For the détermination of the amount, these immunoassaying methods are based on measurements which make it possible to quantify the signais emitted during the analysis of the biological sample. The amount of signais that is detected is generally proportional to the amount, or dose, of analyte to be measured (for example during a sandwich assay) or inversely proportional to the amount, or dose, of analyte to be measured (for example compétition assay).

According to one embodiment, the analysis of the sample of the extraction solution comprising the analytes is carried out by a technique chosen from chemiluminescence, fluorescence, ELISA, EIA, by multiplexing, using biochips, immunochromatographic technique, magnetic beads and radioimmunoanalysis.

Other aims, characteristics and advantages of the invention will emerge in light of the description that will follow and the examples developed hereinafter, which refer to the appended figures wherein:

figures 1 and 2 are diagrammatic représentations of a device 1 which makes it possible to apply the mechanical draining action to the paper according to the method of the invention, and of the general principle for use thereof;

figure 3 is a diagrammatic représentation of another device 2 which also makes it possible to apply the mechanical draining action, and of the general principle for use thereof.

The aim of these examples is to facilitate the understanding of the invention, the implémentation thereof and the use thereof. These examples are given by way of explanation and do not limit the scope of the invention.

The invention will be understood more clearly by means of the following examples which are given by way of nonlimiting illustration.

EXAMPLES

I. Feasibility with the chickenpox virus

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

By way of illustration, the samples were analyzed using the automated immunoanalysis device sold by the company bioMérieux, the VIDAS® System. The VIDAS® VZG (varicella-zoster virus) test is an aid to the diagnosis of a chickenpox virus infection; it makes it possible to detect the immunoglobulin G.

VIDAS® is an automated multiparametric immunoanalysis device. It is a closed system for unit tests, offering great flexibility. This automated device is characterized by its robustness, its flexibility and its ease of use and is intended for small and medium-sized laboratories. It makes it possible to carry out routine tests, confirmation tests and tests with a high medical value.

The détection is carried out via the ELFA (Enzyme Linked Fluorescent Assay) technique in sérum or plasma. The principle of the ELFA assay corresponds to the combination of immunoenzymatic reactions with end-point détection by fluorescence. The enzyme used is alkaline phosphatase which catalyzes the reaction for hydrolysis of the substrate, 4-methylumbelliferyl phosphate to a product: 4-methylumbelliferone. The product emits at a wavelength of 450 nm after excitation at 370 nm. The results are automatically analyzed by VIDAS® and expressed as relative fluorescence intensity or RFV (relative fluorescent value). This RFV value is determined by subtracting the value of the background noise (BKG) from the raw value obtained.

Firstly, the characteristics of the paper and of the extraction solution required for the method according to the invention to be carried out correctly were studied.

In this part, the objective will be the dissolving ofthe analytes of interest in the extraction solution for the implémentation in a VIDAS® VZG test.

In order to make it possible to implement it under conditions where taking the sample, storage, transportation and, finally, analysis are difficult, this method according to the invention must be easy to carry out and ergonomie, and must make it possible to render a resuit preserving the clinical status (positive, négative or equivocal) ofthe sample.

a) Absorption capacity of the papers

903 paper from Whatman® is conventionally used during application on blotting paper, in the context of the DBS technique, in molecular biology. This very fine paper is suitable for handling one to three drops of blood. In this situation, a small amount is sufficient since the nue? aNds to Ν- sought are subjected to a PCR amplification.

ogical diagnosis using an automated immunoanalysis device, it is iiv ;O work with a paper which makes it possible to deposit and recover a larger volume of blood sample. The VIDAS® tests, for example, require 100 μΐ of sérum for the test on the hepatitis C virus (HCV), 150 μΐ for the test on the hepatitis B virus (HBV) and 200 μΙ ’ip A OS virus (HIV). These volumes are substantially équivalent for the other ivau -esent on the market.

Thus, the pa_t ust therefore be capable of absorbing but also returning a sr''~ ient volume of sample, immediately after drying or after a storage time.

In order to select the paper most suitable for the method, a study is carried out on 6 types of paper supplied by the company Ahlstrom® (TNF, 226, 222, 350, 270 and 320). These papers are characterized by their basis weight, their weight and their thickness. The TNF and 226 papers hâve characteristics similar to the 903 paper from Whatman® which is conventionally used in the context of the DBS technique.

Référencé (Grade)	Composition	Basis weight (g/m²)	Thickness (mm)
167L	Cotton	360	1
226	Cotton	179	0.52
222	Cotton	291.3	0.82
270	Cotton	438.8	1.81
320	Cotton	702	2.47
350	Cotton	224.6	1.39
TNF	Cotton	179	0.48

Table 1: Characteristics ofthe papers and schematic représentation of the thicknesses

The paper must hâve certain characteristics. Indeed, it must hâve a fiat and uniform surface, allowing uniform diffusion of the blood sample over its entire surface at the time of the deposit.

Furthermore, the paper must be easy to handle, with a reasonable drying time estimated at two hours, and therefore must not hâve too large a thickness.

The diameter of the disks is an important factor. Each paper has a limiting volume not to be exceeded because, above it, the paper no longer absorbs the sample. It is therefore necessary to precisely define the diameter of the disks. The table below represents the maximum absorbable volume for the papers of grades 222 and 270.

Paper grade	222	270
Disk diameter (mm)	20	19.5	20	19.5
Volume of sérum deposited (μΐ)	300	250	500	450

Table 2: Volume absorbable by the papers as a function of the diameter

The results show that a variation of 0.5 mm regarding the diameter results in a controlled absorption capacity decrease. The volume deposited must impregnate the whole of the paper but without overflowing.

For the subséquent experiments, a paper in disk form 20 mm in diameter which makes it possible to deposit above 200 μΐ of blood sample will be investigated. This is because the handling of disks of a higher diameter, 30 to 50 mm, can quickly become difficult. Thus, the blotting paper according to the invention must hâve an absorption capacity of at least 60 pl.cm’².

The various papers mentioned in table l are tested with three different sérum volumes: 100, 200 and 500 μΐ. These volumes can correspond to the sample test specimens required for a test on an automated immunoanalysis device.

Reference (Grade)	100 μΐ	200 μΐ	500 μΐ
270	11	14	18
320	12	15	19
350	13	17	25
167L	13	17	26
222	14	19	29
226	16	24	36
TNF	16	23	37

Table 3: Diffusion diameter (mm) according to the paper and the volume deposited

After deposit of the sérum, the diffusion diameter for each paper was measured. Given the diameter chosen for the paper, above 20 mm of diffusion diameter, it is considered that the paper is no longer capable of absorbing the sérum. In other words, the paper must hâve a capacity for absorption of at least 60 pl.cm’² of a blood sample.

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

b) Extraction solution

The extraction solution which allows the recovery of the analytes that were absorbed into the fibers of the blotting paper during the drying was then studied.

As previously indicated, this solution has a dual function, namely to allow the extraction of the analytes initially présent in the starting sample without denaturing them and to mimic the blood matrix of the initial sample so as to allow the analysis thereof on an automated immunoanalysis device, this altogether making it possible to preserve the clinical status of the starting sample.

Thus, in the context of the invention, an extraction solution of simple formulation, as physiological as possible, which does not dégradé the trapped analytes, is sought.

Ten extraction solution formulations are tested. Firstly, the impact of these various solutions on a sérum with démonstration of their performance quality of a nontoxic diluent on the structure of the analytes is observed.

The study of the nontoxicity of the extraction solution is carried out by means of tests on pure sera and sera diluted 50/50 in each extraction solution. The nontoxicity of a solution is confirmed if the ratio of the signal in RFV of the pure sérum to that of the sérum diluted 50/50 is less than two.

These extraction solutions are tested on various samples of sérum having different amounts of analytes. The table below represents the signal obtained on VIDAS® for each sample of pure sérum and diluted sérum in an extraction solution.

	Sérum l (> 500 RFV)	Sérum 2 (> 500 RFV)	Sérum 3 (> 1000 RFV)
l	l/2	(pure serum/diluted sérum) ratio	l	l/2	(pure serum/diluted sérum) ratio	l	l/2	(pure serum/diluted sérum) ratio
PBS 50mMNaCI !54mM \| Tween20 0.05% BSA 5%	758	495	1.53	695	401	I.73	1003	810	1.24
PBS 50mM NaCl !54mM	758	555	1.37	695	515	1.35	1003	940	l .07
PBS 50mM NaCl !54mM \| Urea l M BSA 5%	758	476	1.59	695	396	1.76	1003	751	1.34

PBS 50mM NaCI 308 mM Tween20 0.05% BSA 5%	758	459	1.65	695	435	1.60	1003	784	1.28
PBS 50mM NaCI !54mM Tween20 0.5% BSA 5%	758	524	1.45	695	414	1.68	1003	799	1.26
PBS 50mM NaCI !54mM Tween20 5% BSA 5%	758	505	1.50	695	407	1.71	1003	805	1.25
PBS lOmMNaCl !54mM Tween20 0.05% BSA 5%	758	503	l.5l	695	389	1.79	1003	779	1.29
PBS !50mMNaCl I54 mM Tween20 0.05% BSA 5%	758	469	1.62	695	398	1.75	1003	853	1.18
Tris lOmM NaCI 154 mM Régilait 0.3%	758	421	1.80	695	358	1.94	1003	764	1.31
PBS 50mM NaCI l54mM Tween20 0.05%	758	401	1.89	695	377	1.84	1003	751	1.34

	Sérum 4 (> 1000 RFV)
1	1/2	(pure serum/diluted sérum) ratio
PBS 50mM NaCI 154mM Tween20 0.05% BSA 5%	1019	598	1.70 '
PBS 50mM NaCI 154mM Triton 1% BSA 5%	1019	673	1.51
PBS 50mM NaCI 154mM Urea 1MBSA5%	1019	621	1.64
PBS 50mM NaCI 308 mM Tween20 0.05% BSA 5%	1019	590	1.73
PBS 50mM NaCI 154 mM Tween20 0.5% BSA 5%	1019	647	1.57
PBS 50mM NaCI 154 mM Tween20 5% BSA 5%	1019	671	1.52
PBS lOmMNaCl 154 mM Tween20 0.05% BSA 5%	1019	625	1.63
PBS 150mMNaCI 154 mM Tween20 0.05% BSA 5%	1019	573	1.78
Tris lOmMNaCl 154 mM Régilait 0.3%	1019	625	1.63
PBS 50mMNaCl 154 mM Tween20 0.05%	1019	549	1.86

Table 4: Composition and efficiency of the extraction buffers

In the light of the aforementioned, the extraction solutions hâve no toxic effect on the sera. Thus, these solutions do not dénaturé the analytes contained in the sample, while at the same time reproducing physiological conditions thus allowing their analysis on automated analysis devices.

c) Extraction devices

With the aim of solving the technical problem, namely extracting the analytes absorbed into a blotting paper without denaturing them but while at the same time preserving the clinical status of the starting sample, the method of the invention comprises a step of mechanical draining action.

In order to illustrate the invention, and only by way of example, two devices are used to apply the mechanical action to the paper according to the method of the invention.

The two devices based on an extraction by pressure make it possible to recover a sufficient volume for a VIDAS® test.

The first device, represented in figure 1, comprises a lever 1 and an element acting as a piston 2, imperméable to liquids and providing a seal with the side walls of a container 3, which is capable of cooperating with said container 3.

As represented in figure 1, the actuation of the lever 1 allows the element acting as a piston 2 to apply a mechanical draining action on the blotting paper 3 impregnated with the extraction solution in the container 4. The extraction solution 5 resulting from the draining action comprising the analytes contained in the initial blood sample is recovered in a réceptacle 6.

The second device, represented in figures 2 and 3, comprises:

- a container 7 suitable for containing at least one blotting paper 8 and a liquid solution, said container having a fiat bottom comprising a vent hole 9 located on its lower part in order to let the extraction solution 14 pass through,

- an upper element 10 acting as a piston, imperméable to liquids and providing a seal with the side walls of the container 7, comprising a clamping handle 11 and provided with a screw thread 12 suitable for cooperating with the container 7 by screwing, and

- a lower element 13 which makes it possible to recover the extraction solution 14 comprising the analytes contained in the initial blood sample.

As represented in figure 3A, the container 7, comprising the blotting paper 8 and the extraction solution 14, coopérâtes, firstly, with the upper element 10 acting as extraction 14. Once these three éléments are assembled, the clamping handle 11 is lowered, as illustrated in figure 3B, so as to allow the screwing of the upper element 10 with the container 7 and thus multiplying the force applied. Figure 3C shows the extraction solution 14 recovered following the mechanical draining action applied to the blotting paper 8 by the upper element 10.

The two devices based on an extraction by pressure make it possible to recover a volume, greater than or equal to 100 μΐ, sufficient for a VIDAS® test. The first System, that will subsequently be referred to as Device 1, is a vertical clamping toggle making it possible to apply a uniform pressure of 100 N, and makes it possible to recover at least 70% of the volume of solution deposited, that is to say more than 400 μΐ extracted for 600 μΐ deposited. It allows a reproducible extraction since it opérâtes independently of the strength of the user. The second System, that will subsequently be referred to as Device 2, for its part makes it possible to recover at least 50% of the volumes.

piston and, secondly, with the lower element 13 making it possible to recover the solution

	Device 1	Device 2
Volume recovered (μΐ) Ratio	Volume recovered (μΐ)	Ratio
Volume deposited 600 μΐ	424 \| 0.71	322	0.54

Table 5: Volume of extraction solution recovered as a function ofthe device used

The Device 1 and the papers, having a diameter of 20 mm, of grades 222, 350 and 270, will be used for the subséquent experiment.

The sérum sample from a patient séropositive for chickenpox, in other words the clinical status of which is positive for chickenpox, is tested firstly on the VIDAS® in order to establish the value of its signal. It will subsequently be referred to as initial signal. Secondly, the step of depositing and drying on paper will be carried out according to the invention before carrying out the extraction step, using the extraction solution, the volume of which is équivalent to the volume of sample deposited, and the Device 1, followed by the VIDAS® test. The resuit given by the VIDAS® will be the signal recovered. The value of the initial signal and those of the signais recovered, in RFV, the clinical status and the signal percentage are presented in the table below.

	Test for recovery of the signal and préservation ofthe clinical status
Paper grade	222	350	270
Drying time	1 h 30	2h	2h
Drying température	25°C
Extraction solution	50 mM PBS 154 mM NaCl 0.05% Tween 20
Volume of sérum deposited 2 disks (μΐ)	600	700	1000
Volume of extraction solution 2 disks (μΐ)	600	700	1000
Re-impregnation time (min)	2
Initial signal (RFV)	Initial clinical status	1428	positive	1428	positive	1428	positive
RFV recovered (RFV)	Clinical status recovered	1413	positive	1195	positive	1195	positive
Signal recovered (%)	98.95	83.68	83.68

Table 6: Test for recovery of the signal and préservation of the clinical status

The results show that the sample extracted, using the extraction solution and the device 1, is in a volume sufficient to enable the VIDAS® VZG test. Without dilution of the sample, that is to say that the initial volume of sample is exactly the same as the volume of extraction solution (volume deposited = volume of extraction solution), the signal recovered remains high. Indeed, it remains greater than 80% ofthe initial signal.

Furthermore, the clinical status of the sample recovered is the same as that of the initial sample, that is to say positive. In other words, the sample recovered following the extraction step remains séropositive with respect to chickenpox just like the initial sample. Thus, the clinical status is well preserved.

These results also show that a time oftwo minutes for re-impregnation ofthe paper with the extraction solution is sufficient to obtain a satisfactory signal (greater than 80%). This time allows the extraction solution to diffuse into the fibers of the paper which is thus better rehydrated, thereby improving the release of the trapped analytes.

The varions assays carried out in parallel with the other extraction solutions, mentioned in table 4, show that they also make it possible to obtain a signal greater than 80% of the initial signal (not shown).

Consequently, the method of the invention makes it possible to preserve the clinical status of an initial sample in the context of the use of the DBS technique on an automated immunoanalysis device. Indeed, despite the high volumes deposited on blotting paper (300, 350 and 500 μΐ), 80% of the analytes initially contained in the starting sample are présent in the samples recovered, thereby making it possible to preserve the clinical status of the initial sample.

With the aim of showing the reproducibility of the method according to the invention, the inventors hâve proved that it also opérâtes in the context of détections of other 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 to the HIV test

With the aim of showing the reproducibility of the method, the diagnosis of the AIDS virus is studied.

The human immunodeficiency viruses (HIVl and HIV2) are RNA retroviruses. The infection is an infection of the CD4⁺ cells (T lymphocytes, macrophages).

The diagnosis of an infection with this virus is based on the simultaneous détection of the p24 antigen of HIV-l, présent for the first three weeks between the time of the contamination and the appearance of the first antibodies, and the détection of anti-HIV-l and anti-HIV-2 antibodies, which allows monitoring of the séroconversion window.

In the light of the preceding results, the method according to the invention allows diagnosis for clearly positive samples. Indeed, with a method which makes it possible to recover 60-70% of extracted sample and more than 80% of RFV response, the risk of a change in clinical interprétation of the status from positive to négative is unlikely. The situation is different for the weakly positive and equivocal samples which are more readily susceptible to a change in clinical status, which is the case in the context of a diagnosis of the AIDS virus.

By way of illustration, analyses of the samples using the automated immunoanalysis device sold by the company bioMérieux, the VIDAS System, are carried out. The VIDAS® HIV DUO Quick kit, which enables the diagnosis of this virus, requires a large sample test specimen (200 μΐ) and is suitable for the détection of very small amounts of analytes.

The aim is to be able to screen for the infection as close as possible to the contamination, thus when the antibody titer is still low. For the VIDAS® HIV DUO Quick kit, the cutoff which corresponds to the positivity threshold is 125 RFV. In other words, a sample having a cutoff greater than 125 RFV is diagnosed as being séropositive for the AIDS virus.

For this experiment, 500 μΐ of sera are deposited on paper 20 mm in diameter and of grade 222 and dried for 1 h 30 at 30°C. For the extraction step, the paper is left to impregnate in 500 μΐ of extraction solution (50 mM PBS, 154 mM NaCl, 0.05% Tween 20) for a period of 2 minutes. The two devices are used for the step of mechanical draining action on the paper.

The sérum sample used for the experiment is séropositive for HIV. The value of the initial signal and those of the recovered signais, in RFV, the clinical status and the signal percentage are presented in the table below.

	Device 1	Device 2
Initial 1 Initial signal clinical (RFV) 1 status	196	Positive	196	Positive	196	Positive	196	Positive
Recovered \| Recovered RFV clinical (RFV) 1 status	148	Positive	147	Positive	138	Positive	134	Positive
Recovered signal (%)	76	75	70	67

Table 7: Study of the détection limit for HIV

The results show that the recovered signal is around 70% after use of the method according to the invention. The recovered signal is less than that obtained in VIDAS VZG, nevertheless, it still makes it possible to preserve the clinical status of origin. Indeed, the samples recovered following the extraction step remain séropositive for HIV.

ΠΙ. Transposition ofthe method according to the invention to the HBV and HCV tests

The hepatitis B virus (HBV) is a DNA virus composed of an outer lipid envelope and a protein nucleocapsid containing the viral DNA. The envelope contains HBs proteins which are anchored and exposed at the surface of the virus in order to enable the steps of binding to and pénétration into hépatocytes.

Following an infection with the HBV virus, the HBs antigen (HBsAg) appears after several days and can persist for several months (= chronic infection). Usually, the disappearance of HBsAg is followed by the appearance of anti-HBs antibodies. The search for the antibodies is carried out during infected patient follow-ups, but also to verify HBV vaccination efficacy.

The sample analyses are carried out using the automated immunoanalysis device sold by the company bioMérieux, the VIDAS® system, and the VIDAS® HBsAg test.

For a détection of hepatitis B virus HBs antigens with the VIDAS® HBsAg kit, from 150 μΐ of sérum are required. The test used is the VIDAS® HBsAg Ultra kit, sold by the company bioMérieux, which defines a positivity threshold index at 80 RFV. The threshold is thus very low in the VIDAS® RFV reading scale.

500 μΐ of sera are deposited on paper 20 mm in diameter and of grade 222 and dried for l h 30 at 30°C. The paper imprégnation step is carried out in 500 μΐ of extraction solution (50 mM PBS, 154 mM NaCl, 0.05% Tween 20) for a period of 2 minutes. The devices l and 2 are used for the step of mechanical draining action on the paper.

The sérum sample used for the experiment is séropositive for the hepatitis B virus. The value ofthe initial signal and those ofthe recovered signais, in RFV, the clinical status and the signal percentage are presented in the table below.

	Device l	Device 2
Initial signal (RFV)	Initial clinical status	H7	Positive	H7	Positive	H7	Positive	H7	P
Recovered RFV (RFV)	Recovered clinical status	98	Positive	96	Positive	97	Positive	93	P
Recovered signal (%)	84	82	83	79

Table 8: Study of the détection limit for HBV

The results show that, on a sérum sample characterized as being close to the positivity threshold, the clinical status remains positive for the HBV virus in ail situations.

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

The hepatitis C virus (HCV), discovered by molecular biology methods, affects ail countries. The HCV infection often progresses to chronic hepatitis C (approximately 80%), exposing the infected individual to the risks of liver complications. The VIDAS® Anti-HCV kit, sold by bioMérieux, is a qualitative test which allows the détection of immunoglobulin G antibodies directed against this virus. For this test, the positivity threshold index is very high, it is defined at 1745 RFV.

For this experiment, 500 μΙ of sera are deposited on paper 20 mm in diameter and of grade 222 and dried for l h 30 at 30°C. For the extraction step, the paper is left to impregnate in 500 μΐ of extraction solution (50 mM PBS, 154 mM NaCl, 0.05% Tween 20) of a period of 2 minutes. The two devices are used for the step of mechanical draining action on the paper.

The sérum sample used for the experiment is séropositive for the hepatitis C virus.

The value of the initial signal and those of the recovered signais, in RFV, the clinical status and the signal percentage are presented in the table below.

	Device l	Device 2
Initial \| Initial signal clinical (RFV) status	1921	P	1921	P	1921	P
Recovered Recovered RFV clinical (RFV) I status	1898	P	1856	P	1978	P
Recovered signal (%)	99	97	91	103

Table 9: Study of the détection limit for HCV

In this situation, the cutoff is very high (1745 RFV); nevertheless, it is possible to observe that the status remains identical between the initial sample and the recovered sample following the method according to the invention. The latter is thus applicable.

Claims

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

blotting paper is provided on which a blood sample, with an initial volume of greater than or equal to 100 μΐ, is dried, said blotting paper having a capacity for absorption of said sample of at least 60 pl.cm'² and a basis weight of between 200 g.m'² and 800 g.m^-2;

- said blotting paper is transferred into a container;

- a liquid extraction solution is added to said container, said extraction solution being chosen from:

o a phosphate-buffered saline having a phosphate concentration of between 10 mmol.l·' and 150 mmol.l·¹ and a sodium chloride concentration of between 150 mmol.l·¹ and 350 mmol.l·¹ and o a Tris-buffered saline having a Tris concentration of between 10 mmol.l·¹and 150 mmol.l·¹ and a sodium chloride concentration of between 150 mmol.l·¹ and 350 mmol.l·¹, said extraction solution having a volume équivalent to the volume of blood sample dried on the blotting paper;

- said blotting paper is left to impregnate in the extraction solution;

a mechanical draining action is applied to said blotting paper; and the extraction solution comprising the analytes contained in the initial blood sample is recovered.

2. The method as claimed in 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 as claimed in claim 1 or 2, wherein the extraction solution also comprises détergents and/or carrier proteins and/or ion-chelating agents and/or milk powder.

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

5. The method as claimed in any one of claims l to 4, wherein the blotting paper is a nonwoven fibrous material comprising fibers chosen from cellulose fibers and glass fibers.

6. The method as claimed in any one of claims l to 5, wherein the blotting paper has a thickness of between 0.8 mm and 2,50 mm.

7. The method as claimed in any one of claims l to 6, wherein the blotting paper is in the 10 form of a disk.

8. The method as claimed in any one of claims 1 to 7, wherein the mechanical draining action on the blotting paper is carried out by manual mechanical pressure.

15 9. The method as claimed in any one of claims 1 to 8, wherein the container is capable of cooperating with a means sliding inside said container, said means being imperméable to liquids and providing a seal with the side walls of the container.