WO2012143894A1

WO2012143894A1 - Method and device for the determination of analytes in whole blood

Info

Publication number: WO2012143894A1
Application number: PCT/IB2012/051984
Authority: WO
Inventors: Gianfranco Liguri
Original assignee: Gianfranco Liguri
Priority date: 2011-04-20
Filing date: 2012-04-19
Publication date: 2012-10-26
Also published as: ITFI20110078A1

Abstract

The invention relates to a method for the determination of analytes in plasma obtained from venous or capillary whole blood, including filtration of diluted whole blood, comprising anticoagulant and photometric traces, through a microporous membrane (4); and to a device for carrying out such a method.

Description

Method and device for the determination of analytes in whole blood

Field of the Invention

[1] The presently described invention relates to a method and a device for determining analytes in diluted plasma obtained by separating formed elements of venous or capillary whole blood

Background Art

[2] Human blood has a highly complex composition. On the other hand, its analysis is the simplest and effective way for identification and monitoring of various pathologies. Blood samples taken from patients for the analyses consist of the so-called 'whole blood', in which a corpusculated fraction is suspended in plasma, which is approximately 55% of the total volume. The remaining volume is occupied by formed elements that include red blood cells, white blood cells and platelets. The plasma is composed of approximately 90% water and the remainder of metabolites, hormones, inorganic electrolytes and proteins including albumin, globulins, fibrinogen, thrombin and other clotting factors.

[3] To date, the methods commonly employed for the separation of the formed

elements of the plasma are based on centrifugation or filtration. In clinical practice, venous blood samples are collected from patients by venipuncture into tubes containing anticoagulant, where, by centrifugation, the separation of plasma from the formed elements can be obtained. Alternatively, the blood is coagulated and the serum is separated from the clot. The plasma or serum thus obtained are then used to perform biochemical, immunochemical and other kind of analyses, to detect and quantify metabolites, hormones, and other components.

[4] Centrifugation is the method used currently for the separation of whole blood into plasma and formed elements, when traditional methods of analysis are used. In these cases, the volume of venous blood collected with the appropriate syringe varies between one and several milliliters, volumes of plasma necessary for each individual laboratory analysis being typically comprised between 5 and hundreds of microliters.

[5] The centrifuge has, however, some disadvantages that affect the use. In the first place a method is not rapid, since the complete separation of the two components of the blood requires acceleration of gravity of 1000 - 1500 g, and a time ranging between 10 and 3 minutes. Also by this method is not practically possible to recover the total available volume of the plasma. The physical separation of plasma from the sediment consisting of the particulate component is in fact typically carried out by aspiration or by decantation and is usually stoppeds when about 70% of the plasma has been removed, since the recovery of the remaining volume would entail the risk of partial resuspension of the sediment, in particular of thrombocytes, with consequent contamination of the collected plasma. [6] Even the use of collection tubes with separator gels solves this problem only in part because a significant fraction of plasma, depending on the hematocrit of the sample, remains trapped within the gel. Moreover, even in this case, in the automated systems of separation, the aspiration needle is arrested in a few millimeters from the interface gel / plasma to avoid contamination of the latter with the gel itself. Even in the case in which the blood is allowed to clot in order to obtain the serum for analysis, the cen- trifugation has disadvantages similar to those here described.

[7] Another critical aspect to consider when centrifugation is used to separate plasma from whole blood, is the possible alteration of the composition of the plasma caused from the metabolic activity of blood cells, which remain in contact with the plasma even after the centrifugation stops. A well-known example in this regard is the alteration of blood glucose which, in the absence of inhibitors of glycolysis, tends to decrease even after centrifugation due to erythrocyte metabolism.

[8] This undesirable effect requires the immediate removal of the plasma from the

sediment at the end of centrifugation, or the maintenance at low temperatures (4-5 ⁰ C) of the centrifuged blood to minimize the cellular metabolic activity. However, this drawback does not occur in separation processes based on filtration, since the separation of plasma from the formed elements causes the loss of physical contact between them; filtration presents, however, other disadvantages as explained in more detail here following.

[9] The drawbacks of the centrifugation became much more relevant in the case of capillary blood sampling (by fingerpricking, form ear lobe, from the skin of the foot, etc.). In these cases, blood volumes ranging from a few microliters to several hundred microliters are usually collected. When subjected to centrifugation, volumes of obtained plasma fall very quickly as the sample volume decreases, until it becomes zero when blood volumes approach to few microliters. Additional problems then arise, related to the difficulty of accurately divide the small volumes of plasma into many fractions as the number of tests to be performed on the sample.

[10] Filtration ia another method theoretically applicable for the separation of plasma from whole blood. Indeed, many methods have been proposed, using filter media and different procedures. All the methods proposed so far, however, have the disadvantages of being expensive and time-consuming, as well as to allow the separation of only a small percentage of the available plasma and then to compel the taking of a proportionally greater volume of blood. Furthermore, in certain conditions, the filtration can involve the risk of lysis of red cells (hemolysis) which causes the alteration of the biochemical composition of the plasma, and then the results of laboratory analyses.

[11] These drawbacks are caused mainly by the high viscosity of the blood and by the effect of pores clogging of the of the filter media by the cells of the blood during the filtration process. For these reasons, the filtration has not been established to date as a method of separation of plasma from whole blood in the practice of clinical-chemical analyses.

[12] None of the methods currently employed to separate the whole blood into its

components therefore allows to obtain a volume of the plasma useful to carry out a comprehensive set of biochemical investigations from a sample of capillary blood, typically ranging from few microliters and 200/300 microliters, or from a small sample of venous blood.

[13] It is therefore widely recognized the technical problem of having a method of

separation of the formed elements which allows to obtain by small amounts of blood, such as those typically obtained by lancing from infants or small animals, a volume of plasma sufficient to perform chemical-clinical and immunochemical analyses, and which do not have the drawbacks highlighted above for the known methods.

Summary of the Invention

[14] The Applicant has developed a method and a device for the determination of

analytes in the plasma, obtained in a fast and accurate way by separation of the formed elements of whole blood.

[15] By this method and the relative device is also possible to obtain much larger

amount of plasma compared to known methods, starting from the same quantities of whole blood drawn from the patient. The use of the method of the invention therefore makes it feasible to perform comprehensive analyses on plasma even from small amounts of whole blood obtained from micro-sampling, for example by finger- pricking.

[16] Object of the invention is therefore a method for the determination of analytes in whole blood, venous or capillary, characterized in that it comprises the following steps:

[17] i) dilution of the whole blood sample in an iso-osmolar solution comprising one or more anticoagulants and at least one photometric tracer, said solution having known optical absorbance;

[18] ii) filtration of the diluted whole blood coming from step i), by applying a hydrostatic pressure difference, through a hydrophilic microporous membrane, in which the formed elements of blood are blocked by said membrane while the filtrate, comprising diluted plasma and photometric tracer, passes through the membrane;

[19] iii) recovery of the filtrate coming from step ii) and measuring absorbance of the filtrate by photometry;

[20] iv) determination of the analytes of interest, in which the concentration measured for each analyte is multiplied by a dilution factor Fd calculated according to the following formula:

[21] F_d = (Aj / A_d) / [(Aj / A_d) -1],

[22] where Aj is the absorbance of the photometric tracer in said iso-osmolar solution; and A_d is the absorbance of the tracer in said filtrate, to compute the concentration of the same analytes in the whole blood sample. [23] A further purpose of the invention is a device for implementing said method, characterized by comprising a container adapted to contain a certain volume of iso-osmolar solution and to accomplish the dilution of the whole blood sample; a filter element comprising a microporous membrane hydrophilic and an opening for the outflow of the plasma, and means for generating a hydrostatic pressure difference between the two sides of said membrane. A still further object of the invention is the use of said device for obtaining diluted plasma from capillary or venous whole blood.

[24] Further important features of the method and device of the invention are shown in the following detailed description.

Brief Description of Drawings

[25] Other features and advantages of the invention will become apparent from the following description of embodiments thereof given by way of example and not limited to the accompanying drawings, wherein:

[26] - Figure 1 is a view of longitudinal section of the device of the invention;

[27] - Figure 2 is a view of longitudinal section of the device of Figure 1 in the different configurations assumed during application of the method of the invention, and indicated in the figure by the letters of the alphabet from 'a' through 'g'. A pressure is applied In correspondence of the arrows.

Detailed Description of the Invention

[28] The method of the invention can be applied to any blood sample, both human and animal, for the separation of the formed elements of blood from plasma, and subsequent determination of analytes, using the device shown in the figures.

[29] With reference to Figure 1, the device 1 of the invention comprises at least two components A and B, consisting of plastic, metal, composite or other suitable material, the same or different between them. Component A has the function of container and diluter, and is formed by a container fitted with an opening 2 for the entry of the whole blood to be diluted and inside which a piston 3, at the lower end of the container A, can slide.

[30] The component B functions as the filter element, and comprises a microporous membrane 4, a porous septum 5, one or more gaskets 6 to hermetically seal the area below the membrane 4, and an opening 7 for the outflow of the filtered plasma; the component B is, in size and shape, constructed so as to connect to the upper part of the component A. In Figure 1 is also represented a further component, indicated by C and possibly included in the present device; the component C has the function of cap of the component A, and comprises one or more gaskets 8, which ensure that the component A can be sealed tightly when needed.

[31] The shape and structure of the device as described above can be varied while

remaining within the invention, provided the following items are maintained: two openings, one for the entry of the whole blood to be diluted and one for the exit of the filtrate, a microporous membrane and a container adapted to contain the iso-osmolar solution and to dilute whole blood. The device may, for example, have the configuration of a suitable container with a syringe filter or filter tips from pipette or filters to top.

[32] Alternatively, the components B and C of the device of Figure 1 may be replaced by a cover plate intended to engage with the component A, and fitted with at least one opening for the entrance of the whole blood in the container A and at least a filter element with the same characteristics described above for the component B. Thanks to the possibility of sliding of the cover plate on suitable slithers, possibly controlled by suitable automation systems, the phases of dispensing whole blood in the container A and filtration can be automated.

[33] Figure 2 shows the same device of Figure 1, in different configurations of use and application of the method of the invention, indicated with the letters 'a' through 'g'.

[34] In the configuration 'a', the piston 3 is in the rest position, and the cap C is removed from the vessel A, freeing the opening 2 for the introduction of the iso-osmolar solution 9. Alternatively, the container A can be pre-filled in the production phase, with a given volume of iso-osmolar solution.

[35] In the configuration 'b', the whole blood sample 10 is introduced into the vessel A through the opening 2, and in the configuration 'c', the whole blood is diluted in the iso-osmolar solution to obtain a suspension 11.

[36] The dilution of whole blood (step i) of this process) can be performed, for example, with an amount of iso-osmolar solution such as to achieve a volume ratio between whole blood and iso-osmolar solution falling between 1/1, 2 and 1/120, preferably between 1/5 and 1/30, and more preferably corresponding to 1/20. Preferably the volume of the iso-osmolar solution used is between 100 and 1000 uL.

[37] For 'iso-osmolar ', or 'isotonic' solution is meant a solution of the same osmolarity of the plasma; a solution of sodium chloride in purified water and sterilized, having the same osmolarity of the plasma, can be used for the purpose the invention.

[38] The iso-osmolar solution here used comprises one or more anticoagulants, chosen for example among heparin, sodium etilendiamminotetraacetato (EDTA), sodium citrate, and mixtures thereof; any additional anti-coagulant agent commonly used and suitable to avoid the formation of clots in the plasma can be used the same way.

[39] The iso-osmolar solution according to the invention further comprises at least a suitable photometric tracer, that works as internal reference standard for the analyses of the diluted and filtered plasma; the presence of the photometric tracer allows, in other words, the normalization of the dilution of the blood in a manner not dependent on the volume of the blood itself. Obviously, the photometric tracer that is added to the solution must not have any lysing activity, or otherwise favoring the lysis, of the formed elements of blood, or cause any alterations of their cell membrane which could cause leakage of intracellular material. In addition, tracers suitable for this use should not interfere, nor activate or inhibit, with the complex of biochemical reactions, im- munochemical, genetic or otherwise, that will be carried out on the filtered plasma.

[40] Photometric tracers with a high molar extinction coefficient at one or more

wavelengths of visible light or ultraviolet or infrared light, are particularly suitable for use in the present invention. According to a preferred embodiment of the invention, the tracer has a maximum value of photometric absorption greater than 10² · L · mol ¹ cm ¹ in the region of the electromagnetic spectrum between 560 nm and 2000 nm, preferably between 600 and 740 nm, more preferably between 625 and 700 nm.

[41] A suitable feature for the photometric tracers of the invention is also the stability during time of their absorption spectrum; preferably these tracers have, over 12 months and at the used wavelength, a change in optical absorbance less than 5 %. The nature of the tracer is also such as not to interfere with the biochemical, immunochemical or analysis of other nature to be carried out on the sample.

[42] Photometric tracers suitable for use in the present method can therefore be chosen, for example, among the organic substances used as colored pigments, for the preparation of inks, paints, dyes, and the like, and preferably are selected from the group consisting of methylene blue ( disodium salt of [[4 - [bis [4 - [(sulfonatofenil) amino] phenyl] methylene] cyclohexa-2 ,5-dien-l-ylidene] amino] benzenesulfonic acid, methyl violet (hexamethyl pararosaniline chloride), methylene blue ( 3 , 7bis ( dimethylamino )phenothiazinium chloride ), indigo carmine (disodium salt of 2,2 '-bis (2,3-dihydro-3-oxo-indolen) -5,5' - disulfonic acid), anthocyanins such as anthocyanins extracted from the flowers of the plant called Butterfly pea, and the so-called Brilliant Blue FCF (inner salt, disodium hydroxide). The photometric tracers above are only examples, any substance that responds to the characteristics described above being employable to this end.

[43] The concentration of the tracer in the photometric iso-osmolar solution can vary between 10⁹ M and 10¹ M, and preferably ranges between 10 ⁶ and 10 ³ M.

[44] The iso-osmolar solution of the invention may further comprise one or more

substances capable of causing the agglutination of the erythrocytes, in order to facilitate filtration. Among these substances, preferred are the fitoemoagglutinine, or lectins, such as concanavalin A, and the agglutinating antibodies or agglutinins.

[45] Once whole blood has been diluted with the iso-osmolar solution, it is subjected to filtration (step ii) of this process) through a microporous membrane made from hy- drophilic, or predominantly hydrophilic materials, or otherwise surface-treated so as to be hydrophilic, for example steel or other metallic materials, glass fiber, natural or synthetic fabric, or plastic, for example polyester resins, polymethacrylates, polyamides, polyvinyl pyrrolidone, polysulfone and mixtures thereof. The use in these filtrating membranes of hydrophobic materials, such as polysulfone, will be combined with the use of hydrophilic polymers so that the membrane will turn to be hydrophilic, or with the use of physical treatment such as plasma treatment, in order to impart to the membrane the required hydrophilicity. [46] According to the invention the term 'microporous membrane' means a film of thickness between 0.001 and 5 mm, having pores with an average diameter of micrometer-sized, including for example between 0.1 and 50 μιη; and preferably between 0.2 and 10 μιη. Especially advantageous is the use of membranes with a mean pore diameter between 0.45 and 5 μιη. The term 'microporous membrane' according to the invention is to be understood also inclusive of glass fiber filters, such as those marketed by Pall Gelman with the name of 'glass fiber media'.

[47] According to a preferred embodiment of the present device, the microporous

membrane 3 is also 'asymmetrical', meaning by this term the asymmetry in the distribution of the average size of the pores of the membrane by passing from one side to the other of the membrane itself; as an example, the pores with a larger average diameter, located on the side of the membrane that is in contact with whole blood, have an average diameter between 1 and 50 m m, and preferably between 5 and 20 μιη, whereas the pores with smaller average diameter, which are located on the opposite side, have a diameter between 0.1 and 5 μιη, and preferably between 0.45 and 3 μιη.

[48] Membrane particularly preferred according to the invention is a microporous

membrane consisting of a copolymer asymmetric polysulfone / polyvinylpyrrolidone, such as the membrane described in European Patent EP 0336483.

[49] In Figure 2 the configuration 'd' of the device shows the beginning of the filtration stage of the present method, in which the filtering component B is housed on the container A by exerting a pressure from above up to hermetically seal the space below the membrane, as shown in configuration 'e' in the same Figure 2.

[50] In the device of the invention the filtering component B, in addition to the microporous membrane 4, may comprise the macroporous porous septum 5, which has the function of filter-holder and mechanically supports the membrane when pressure is applied over it. Any solid, macroporous material can be used to prepare such a porous septum suitable for the present device; it can be, for example, a metal mesh, a lattice of plastic material, a sintered glass frit, a fabric, and the like.

[51] The size of the microporous membrane 4 are, for example, between 5 and 500 mm², while those of the porous septum 5 may be the same as those of the membrane or slightly more.

[52] Filtration through the microporous membrane 4 is carried out by exerting a hydrostatic pressure difference between its surfaces. In Figure 2, configuration 'f represents the phase of the method of the invention in which a pressure is exerted on the membrane by means of the piston 3, which is slid along the container A, thus reducing the available internal volume and forcing the diluted blood to pass through the membrane.

[53] In the subsequent configuration 'g' of the same Figure 2, the diluted plasma 12

comes out from the opening 7. Other means, manual or electromechanical, can be used as an alternative to the piston 3 to create the desired hydrostatic pressure difference. At the same purpose, a fluid immiscible with the diluted blood contained into the vessel A may also be introduced in the container, or a vacuum can be applied through the opening 7 to cause the leakage of plasma, or the device may be subjected to cen- trifugation as to generate an appropriate pressure difference between the two sides of the microporous membrane to cause the filtration of the diluted blood through the membrane itself.

[54] The filtrate recovered after passage of the diluted whole blood through the

membrane consists of diluted plasma in which analytes can be determined, both qualitatively and quantitatively, by photometric, fluorometric, or with other known techniques. The concentration of the analytes in the filtered plasma will be calculated taking into account the dilution factor, Fd,. Fd is photometrically determined according to the ratio of dilution of the photometric tracer, which then functions as an internal reference standard. In quantitative terms:

[55] F_d = (Aj / A_d) / [(Aj / A_d) -1],

[56] where Aj = absorbance of the iso-osmotic solution; A_d = absorbance of the diluted plasma after filtration.

[57] It is well known to those skilled in the art that, for the relationship described above to provide a correct value of the dilution factor, the two absorbance values have to be are determined at the same wavelength.

[58] This device can be operated both manually and automatically, by means of drive systems, and automatic control, and will include suitable means for the determination of analytes by known methods for measuring absorbance.

[59] Thanks to the method and device of the invention as described above it is possible to achieve an accurate separation of whole blood into plasma and formed elements, while also limiting the risks of contamination both of the plasma and of the operator who manipulates the samples. Furthermore, due to dilution of the whole blood sample, and the ability to accurately determine the dilution factor by photometry, comprehensive analyses can be carried out and repeated several times on the same sample even from small amounts of blood as those obtained by micro- sampling.

[60] The present method is particularly advantageous when the whole blood sample available for analysis has a volume less than 500 μί, or even less than 100 uL as generally occurs in the case of capillary blood drawing.

[61] The device of the invention is intended to obtain diluted plasma from whole blood, also for uses other than the determination of analytes in the blood.

[62] The following examples are provided for the purpose of illustrating the present invention without, however, representing a significant limitation of the same.

[63] Example 1

[64] A container with the features of the container-diluter 'A' of Figure 1 and having a capacity of 1000 uL has been pre-filled with 600 uL of a solution made up as follows: 20 mM phosphate buffer, pH 7.4; NaCl 128 mM; concanavalin A 1 mg/mL; methyl blue 10 μΜ.

[65] An asymmetric microporous membrane formed by a disc of 10 mm diameter made from polysulfone-polyvinylpyrrolidone copolymer with an average porosity of 2 μιη, produced from Spectral Diagnostics Inc. (Canada), was mounted on a porous septum, so that the side with the larger pores is oriented towards the container-diluter, while the side with the smallest pores is in contact with the porous septum.

[66] Example 2

[67] Comparative tests were conducted, using as a comparison of two known methods of separation, centrifugation and filtration, versus the method object of the present invention, implemented using the device and the iso-osmolar solution described in Example 1.

[68] Volumes variable between 5 and 500 μΐ_^ of capillary blood were subjected to centrifugation at room temperature, using 1 ml 'Eppendorf tubes having a conical base and a 'Stat-spin' centrifuge operating at a gravity acceleration of 1500 g.

[69] Equal volumes of capillary blood from the same individual were subjected to

filtration at room temperature, with a polysulfone -polyvinylpyrrolidone microporous asymmetric filtering membrane having an average diameter of 10 mm and average porosity of 2 μιη. A filtration pressure of 20 mBar was applied.

[70] To evaluate the method of the present invention with respect to the above two

methods of comparison, a variable volume between 5 and 500 μΐ_^ of capillary blood from the same individual was added and mixed with 600 μΐ_^ of iso-osmotic solution contained in the diluter A of the device.

[71] The diluted blood was then filtered at room temperature by applying to the piston

(Item 3 in Figure 1) a force of sufficient strength to obtain a filtration pressure of 20 mBar. The element B of the device was provided with a polysulfone - polyvinylpyrrolidone asymmetric microporous filtering membrane having an average diameter of 10 mm and average porosity of 2 μιη.

[72] In the following Table 1 the second column shows the values of the estimated

volume of plasma in the sample (blood sample volume*hematocrit/100), while the fourth column shows the volume of plasma obtained by the three methods, applied to capillary blood samples collected from the same individual.

[73]

Table 1

Blood volume, Plasma Separation Maximum Time(s) μΐ (hematocrit volume, μΐ method volume of

= 43) available

plasma, μΐ

5 2,15 Centrifugation 0 180 5 2,15 Filtration 0 20

5 2,15 Proposed 1,5 3

method

20 8,6 Centrifugation 3 180

20 8,6 Filtration 0 57

20 8,6 Proposed 6 5

method

50 21,5 Centrifugation 10 180

50 21,5 Filtration 7 83

50 21,5 Proposed 20 10

method

200 86 Centrifugation 50 180

200 86 Filtration 20 114

200 86 Proposed 83 15

method

500 215 Centrifugation 180 180

500 215 Filtration 50 144

500 215 Proposed 210 26

method

[74] Results in Table 1 above show the advantages of the invention as compared to known methods, both in terms of amount of plasma obtained and of time required to get it.

[75] As it can be seen in Table 1, using the known methods no plasma can be obtained when blood amount is equal to 20 μΐ_^ or less.

[76] Example 3

[77] The experimental and comparison tests described in Example 2 were repeated in equivalent conditions, except for the nature of microporous filtration membrane here replaced by a nylon hydrophilic microporous membrane having an average porosity of 0.8 μπι.

[78] Also the filtration pressure was changed, being 40 mBar in this example.

[79] The following Table 2 shows the values obtained by the three methods applied to capillary blood samples drawn from the same individual.

[80]

Table 2

[81] Results from experiments described above in Examples 1 and 2 clearly show that the method of the present invention provides a significantly greater volume of plasma, sufficient for the execution of numerous clinical biochemical tests, when compared to known methods of separating whole blood. Furthermore, separation time is significantly lower with respect to known methods taken here as a comparison. As shown in tables 1 and 2, plasma can not be obtained at all by known methods when volume of blood samples approaches 20 μΐ_^ or below.

[82] Example 4

[83] Glucose and aspartate aminotransferase (AST) assays were carried out using a

Hitachi 911 system on samples of plasma obtained with the three separation methods described above in the Example 2.

[84] The following Table 3 lists the results from quantitative analyses of plasma samples obtained by the three separation methods described above in Example 2, applied to capillary blood samples obtained from five different individuals marked with the letters A, B, C, D and E.

[85]

Table 3

[86] ¹ Samples with marked hemolysis.

[87] ² UI / 1 = International Units per litre.

[88] ³ C.V. = Coefficient of variation, indicates the dispersion of the measures around the mean value. (N= 15): results are from 15 measures.

[89] In the above Table 3: 'NA' means 'not applicable', because the volume of plasma obtained was insufficient to run the analyses; 'Reference method' indicates a traditional method of venipuncture in presence of anticoagulant, plasma separation by cen- trifugation and analysis on Hitachi 911 system. [90] The above-described experiments clearly demonstrate that, when the method of the present invention is applied, accurate and precise results can be achieved even with whole blood volumes less than 50 ul.

[91] On the contrary, it is clear that values of the analytes are inaccurate and imprecise when filtration is used as a method of plasma separation, due to haemolysis, or centrifugation is used, due to the presence of erythrocytes in plasma.

[92] Example 5

[93] On capillary blood samples, 30 μΐ_^ each, separation and plasma recovery have been carried out as described in Example 3 by this method and the two comparison methods filtration and centrifugation. Therefore, the amount of glucose and AST have been determined.

[94] The following Table 4 lists the parameters used to calculate the actual concentration of two analytes, glucose and AST, in the sample, based upon the dilution factor F_d determined as described above. In particular, absorbances at 37° C and 630 nm of the iso-osmolar solution (ABS diluting medium) and of diluted filtered plasma (ABS filtrate) were measured.

[95] 'Measured concentrations' shown in table 4 are the concentrations detected in the plasma, whilst 'calculated concentrations' are concentrations obtained by multiplying the concentrations measured on diluted plasma obtained from this method times the dilution factor F_d .

[96]

Table 4

[97] As indicated in Table 3, even in Table 4 'NA' means 'not applicable', because the volume of plasma obtained was insufficient to run the analysis. As it can be seen in Table 4, a sufficient amount of plasma to perform the analysis was not obtained by traditional filtration. Furthermore, measured analyte concentrations in plasma obtained by centrifugation have proved to be less accurate than those measured in plasma obtained by this method, when compared to the values determined by the standard method, which is defined in the above Example 4.

[98] The examples here described have a mere demonstrative significance and are

neither exhaustive nor comprehensive of this invention.

Claims

[1] A method for the determination of analytes concentration in whole blood,

capillary or venous, comprising the following steps:

i) dilution of said whole blood in a iso-osmolar solution comprising one or more anticoagulants and at least a photometric tracer, said solution having known ab- sorbance at a certain wavelength;

ii) filtration of the diluted whole blood coming from step i) through a hydrophilic microporous membrane, by applying a difference of hydrostatic pressure between the opposite sides of said membrane, wherein the corpuscolar elements of blood are retained by said membrane while the filtrate comprising diluted plasma and the photometric tracer, passes trough the membrane; iii) recovery of the filtrate coming from step ii) and photometric measurement of the filtrate absorbance at the same wavelength of step i);

iv) measurement of the concentration of the analytes of interest in the filtrate coming from step iii) and multiplication of the so measured value of concentration by a dilution factor F_d calculated according to the following formula: F_d = (Ai/A_d)/[(Ai/A_d)- l],

where A_; is the absorbance of the photometric tracer in said iso-osmolar solution of step i); and A_d is the absorbance of the tracer in said filtrate of step iii), in order to obtain the analytes concentration in the staring whole blood.

[2] The method according to claim 1, wherein the dilution of the whole blood in step i) is carried out by adding to the whole blood a volume of said iso-osmolar solution comprised between 100 and 1000 ul, so that a volume ratio between the whole blood and the iso-osmolar solution ranging from 1/1,2 to 1/120, is obtained.

[3] The method according to claim 2, wherein said volume ratio between the whole blood and the iso-osmolar solution ranges from 1/5 to 1/30.

[4] The method according to claim 3, wherein said volume ratio between the whole blood and the iso-osmolar solution is 1/20.

[5] The method according to claim 1, wherein said photometric tracer has a

maximum light absorbance at a wavelength comprised between 560 and 2000 nm, and it is selected from the group consisting of methyl blue, methyl violet, methylene blue, indigo carmine, anthocyanins and Brilliant Blue FCF.

[6] The method according to claim 1, wherein the concentration of said photometric tracer in said iso-osmolar solution ranges between 10⁹ and 10¹ M, and preferably between 10 ⁶ and 10 ³ M.

[7] The method according to claim 1, wherein said hydrophilic microporous

membrane has an average pore diameter ranging between 0,45 and 5 μιη.

[8] The method according to claim 1, wherein said hydrophilic microporous

membrane is asymmetric in the distribution of the average pore diameter on the opposites sides of the membrane.

[9] The method according to claim 1, wherein said hydrophilic microporous

membrane is a membrane consisting essentially of hydrophilic polysulphone, or of an hydrophilic copolymer polyvinylpyrrolidone/polysulphone.

[10] The method according to claim 1, wherein said whole blood has a volume

smaller than 500 ul.

[11] The method according to claim 10, wherein said whole blood has a volume

smaller than 100 uL.

[12] The method according to any of the preceding claims, wherein said whole blood is capillary whole blood.

[13] A device for the determination of analytes concentration in whole blood,

capillary or venous, comprising a container able to contain a certain volume of said whole blood and of an iso-osmolar solution for dilution of the whole blood; a filtration element comprising a hydrophilic microporous membrane and an outlet for the outflow of the filtrate plasma; and means to produce a difference of hydrostatic pressure between the opposite sides of said membrane.

[14] Use of the device as described in claim 13, for the preparation of diluted plasma starting from whole blood, capillary or venous.