WO2012028184A1

WO2012028184A1 - Analysis of blood samples from dried blood spots

Info

Publication number: WO2012028184A1
Application number: PCT/EP2010/062815
Authority: WO
Inventors: Jean-Claude Havaux; Luc Voorn; Vincent Labaye
Original assignee: Zentech S.A.
Priority date: 2010-09-01
Filing date: 2010-09-01
Publication date: 2012-03-08

Abstract

The present invention relates to a method and a device for analyzing a blood sample from a dried blood spot. Said device comprises a first container and a second container. Said method comprises the steps of: providing a dried blood spot on a filter paper; punching a disc of said dried blood spot on said filter paper; placing said disc in said first container; extracting in said first container blood sample from said disc with a liquid solution; inserting said second container in said first container; separating said liquid solution containing blood sample from said disc by removing said liquid solution containing blood sample out of said second container; and analyzing said blood sample; wherein said device permits said liquid solution containing blood sample to be separated from said disc of said dried blood spot. Said device according to the invention for analyzing a blood sample from a dried blood spot on a filter paper disc comprises: a first container, adapted to receive at least one filter paper disc, a liquid solution, and a second container, and a second container disposed with one or more openings, adapted for dismountable insertion at least partially into the first container, whereby, said openings fluidly connect the inside of said first container to the inside of said second container when said second container is inserted into said first container, and said openings are configured to prevent entry of said filter paper disc into said second container.

Description

ANALYSIS OF BLOOD SAMPLES FROM DRIED BLOOD SPOTS

FIELD OF THE INVENTION The present invention relates to a method for analyzing blood samples from dried blood spots. The invention also relates to a device for analyzing a blood sample from a dried blood spot.

BACKGROUND OF THE INVENTION

Dried blood spot or DBS technique consists of blotting few drops of blood onto a filter paper especially designed to absorb blood drops for laboratory analysis. Blood drops are obtained thanks to a light incision carried out to the heel, fingertip or ear lobe. This technique is slightly invasive and generally painless. This is the standard procedure when dealing with diagnosis in neonatology or pediatrics. The use of paper cards, such as Guthrie cards, for the collection of blood samples is particularly suitable for the storage and transport of blood samples at the time of epidemiologic studies or of screening of the population. These cards are usually filter paper made of cellulose. Other well-known advantages of this method are the ease of collection and the short collection time, low sample volume, and sample storage and transport without need for refrigeration.

The biological blood sampling method traditionally used for adults is difficult to apply in neonates and children. In fact, venous sampling is technically difficult in neonates and children, as their veins are tiny and difficult to localize. Further, classic blood drawing is feared by the child and its parents and complicates the work of medical care professionals. Usually, all newborns are routinely screened for certain genetic, metabolic, hormonal and functional disorders. The traditional method for newborn testing starts with the collection of a small amount of blood from newborn within the first postnatal days. The sample is absorbed onto a piece of filter paper to provide a dried blood spot. This sample is sent to the laboratory, where a small disc is punched from the spot and placed into a reaction vessel, wherein the blood is extracted with a liquid solution.

The extraction step causes several drawbacks to the downstream analysis of the blood sample from a dried blood spot. The degradation of the filter paper has been studied. For example, US 5432097 discloses the use of a specific enzyme to digest the filter paper. However, the degradation of the filter paper is partial and the supernatant has to be additionally extracted at least four times from the compacted paper layer. There remains therefore a need for the analysis of a blood sample from a dried blood spot without to resort to filtration or additional extraction steps. The newborn screening tests are routinely performed using an automate platform or manually. The presence of the filter paper disc can make the analysis more difficult by obstructing the needle for automated withdrawal of the blood sample or by interfering with the manual withdrawal of the blood sample. It is therefore an object of the present invention to provide a method to circumvent the presence of the filter paper disc allowing efficient and fast testing of blood samples.

SUMMARY OF THE INVENTION

Surprisingly, the present inventors have found a way to analyze a blood sample from a dried blood spot and overcome the above-mentioned problems of the prior art. In a first aspect, the present invention relates to a method for analyzing a blood sample from a dried blood spot with a device, said device comprising a first container and a second container, said method comprising the steps of: providing a dried blood spot on a filter paper; punching a disc of said dried blood spot on said filter paper; placing said disc in said first container; extracting in said first container blood sample from said disc with a liquid solution; inserting said second container in said first container; separating said liquid solution containing blood sample from said disc by removing said liquid solution containing blood sample out of said second container; and analyzing said blood sample; wherein said device permits said liquid solution containing blood sample to be separated from said disc of said dried blood spot.

The inventors have surprisingly found that the above-provided method separates the liquid containing the blood sample from the disc punched from a dried blood spot. This separation is advantageous because the filter paper disc does not interfere with the downstream analysis of the blood sample obtained from the dried blood spot. Furthermore, this is advantageous because the method obviates the need for filtration or additional extraction steps. Advantageously, the analysis of the blood sample can be performed in an efficient and hence economical way. In a second aspect, the present invention relates to a device for analyzing a blood sample from a dried blood spot on a filter paper disc comprising: a first container, adapted to receive at least one filter paper disc, a liquid solution, and a second container, and a second container disposed with one or more openings, adapted for dismountable insertion at least partially into the first container, whereby, said openings fluidly connect the inside of said first container to the inside of said second container when said second container is inserted into said first container, and said openings are configured to prevent entry of said filter paper disc into said second container.

The present invention is advantageous, because a device as described above separates the liquid containing the blood sample from the filter paper disc punched from a dried blood spot. Advantageously, the present invention provides a device that avoids the interference of the filter paper with the downstream analysis of the blood sample. This is advantageous because the present invention provides a device that allows efficient and fast testing of blood samples both manually and in an automated way, thereby decreasing the cost of the analysis.

The present invention will now be further described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

DETAILED DESCRIPTION OF THE INVENTION

In the following passages, different aspects of the invention are described in more detail. Each aspect so described may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous. In the context of the present invention, the terms used are to be construed in accordance with the following definitions, unless a context dictates otherwise.

As used herein, the singular forms "a", "an", and "the" include both singular and plural referents unless the context clearly dictates otherwise. The terms "comprising", "comprises" and "comprised of" as used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

The term "about" as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of +/-10% or less, preferably +/-5% or less, more preferably or less, and still more preferably +/-0.1 % or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier "about" refers is itself also specifically, and preferably, disclosed.

The present invention relates to a method for analyzing blood samples from dried blood spots. In particular, the invention is directed to a method for analyzing blood samples from dried blood spots, wherein the liquid containing the blood sample is separated from the disc punched from a dried blood spot. The invention further relates to a device for analyzing a blood sample from a dried blood spot on a filter paper disc.

The present invention provides a method for analyzing a blood sample from a dried blood spot with a device, said device comprising a first container and a second container, said method comprising the steps of: providing a dried blood spot on a filter paper; punching a disc of said dried blood spot on said filter paper; placing said disc in said first container; extracting in said first container blood sample from said disc with a liquid solution; inserting said second container in said first container; separating said liquid solution containing blood sample from said disc by removing part of said liquid solution containing blood sample out of said second container; and analyzing said blood sample; wherein said device permits said liquid solution containing blood sample to be separated from said disc of said dried blood spot.

In an embodiment, blood of neonates is collected between the third and the fifth day of life (48 to 120 hours after birth). The infant's heel is punctured with a sterile blood lancet. The blood drop obtained is soaked onto the filter paper in the centre of the circle printed on the specimen collection paper. Both sides of the paper have to be completely saturated with blood. The sample is then air-dried for 4 hours at room temperature and stored in sealed paper envelopes or containers that will provide protection from moisture, light, heat and contact with other materials. The sample discs should be punched from similar areas on each individual blood spot. The dried blood samples can be stored at 2-8°C for a period of at least 4 months.

In an embodiment, the invention provides a method, wherein said first container is a reaction vessel and said second container is a reaction vessel with at least one opening and wherein the dimension of said opening is smaller than the dimension of said disc of a dried blood spot. In a particular embodiment, said second container is a reaction vessel with at least one round opening and wherein the diameter of said round opening is smaller than the diameter of said disc of a dried blood spot.

Preferably, the diameter of said round opening may be ranging between 1.0 and 4.8 mm. More preferably, the diameter of said round opening is 1 .0, 1 .1 , 1.2, 1.3, 1 .4, 1 .5, 1.6, 1.7, 1 .8, 1.9, 2.0, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1 , 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7 or 4.8 mm. Preferably, the diameter of said disc of a dried blood spot may be ranging between 2.0 and 5.0 mm. More preferably, the diameter of said disc is 2.0, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1 , 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or 5.0 mm.

In an embodiment, the extraction of the blood sample from the disc with a liquid solution in said first container is preferably done by agitation. The agitation is done preferably at a speed value ranging from 300 to 900 rpm and can be done using conventional shakers or agitators known in the art, capable of agitating a fluid containing container, reservoir, recipient, plate, reaction vessel, tube, Eppendorf™ tube or bottle.

In an embodiment, the invention relates to a method, wherein said liquid solution reaches a pH value ranging from 2.0 to 13.0. Preferably, the pH value of said liquid solution may range from 6.0 to 8.5. More preferably, the pH value of said liquid solution may be 6.0, 6.5, 7.0, 7.5, 8.0 or 8.5, or a value in the range between any two of the aforementioned values.

In another embodiment said liquid solution may be an aqueous solution, an aqueous acidic solution, phosphate buffered saline (PBS), Tris buffered saline (TBS), salt or detergent. In another embodiment, said disc may be extracted in an aqueous solution of at least 0.05 molar containing hydrochloric acid, acetic acid, trichloroacetic acid (TCA), PBS or TBS. In a preferred embodiment, said disc may be extracted in an aqueous solution having a concentration ranging from 0.05 to 5.0 molar and containing hydrochloric acid, acetic acid, TCA, PBS or TBS. Preferably, said disc may be extracted in an aqueous solution having a concentration ranging from 0.1 to 1.0 molar and containing hydrochloric acid, acetic acid, TCA, PBS or TBS. In particular, said disc may be extracted in an aqueous solution having a concentration of 0.1 , 0.5 or 1 .0 molar, or a value in the range between any two of the aforementioned values, and containing hydrochloric acid, acetic acid, TCA, PBS or TBS.

In another embodiment, the extraction of said disc may occur at a temperature ranging from 20°C to 37°C. Preferably, the extraction of said disc may occur at a temperature of 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36 or 37°C, or a value in the range between any two of the aforementioned values.

In another embodiment, the extraction of said disc may be performed for 1 to 120 minutes. Preferably, said extraction of said disc may be performed for 1 to 90 minutes, more preferably for 1 to 60 minutes. In a preferred embodiment, said extraction of said disc may be performed for 1 , 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 minutes, or a value in the range between any two of the aforementioned values.

In another embodiment, the invention provides a method, wherein said liquid solution containing said blood sample provided as defined above contains lysed blood cells. In an embodiment, the invention provides a method, wherein said liquid solution containing blood sample provided as defined above is analyzed to diagnose neonatal or pediatric disorders.

In a further embodiment, the invention relates to a method, wherein said liquid solution containing blood sample provided as defined above is analyzed to diagnose inborn errors of metabolism, such as disorders of carbohydrate metabolism, disorders of amino acid and organic acid metabolism, disorders of fatty acid oxidation and mitochondrial metabolism, disorders of porphyrin metabolism, disorders of purine or pyrimidine metabolism, disorders of steroid metabolism, disorders of mitochondrial function, disorders of peroxisomal function. Non limiting examples of "disorders of carbohydrate metabolism" may include galactosemia, glycogen storage disease, lactose intolerance, fructose intolerance (aldolase B deficiency), fructosuria (hepatic fructokinase deficiency), galactose epimerase deficiency, galactokinase deficiency or hyperglycerolemia (glycerol kinase deficiency).

Non limiting examples of "disorders of amino acid metabolism and organic acid metabolism" may include phenylketonuria (PKU), tyrosinemia (type I, II or III), alcaptonuria, homocystinuria, histidinemia, maple syrup urine disease (MSUD type lb or II), methylmalonic aciduria, non-ketonic hyperglycinemia type I (NKHI), hyperlysinemia, carbamyl phosphate synthetase I deficiency, ornithine transcarbamylase deficiency, N- acetylglutamate synthetase deficiency, argininosuccinic aciduria, hyperargininemia, citrullinemia, ornithine aminotransferase deficiency, cystinuria (type I or II), hartnup disease or hyperammonemia-hyperornithinemia-homocitrullinuroa syndrome. Non limiting examples of "disorders of fatty acid oxidation and mitochondrial metabolism" may include very-long-chain acyl-CoA dehydrogenase deficiency (VLCAD), long-chain acyl-CoA dehydrogenase deficiency (LCAD), medium-chain acyl-CoA dehydrogenase deficiency (MCAD), short-chain acyl-CoA dehydrogenase deficiency (SCAD), carnitine translocase deficiency, carnitine palmitoyltransferase I (CPT I) deficiency or carnitine palmitoylransferase II (CPT II) deficiency.

Non limiting examples of "disorders of porphyrin metabolism" may include acute intermittent porphyria, congenital erythropoietic porphyria (CEP), erythropoietic protoporphyria (EPP), ALA dehydratase deficiency porphyria (ADP), hereditary coproporphyria (HCP), variegate porphyria (VP), porphyria cutanea tarda (PCT) or hepatoerythropoietic porphyria (HEP).

Non limiting examples of "disorders of purine or pyrimidine metabolism" may include Lesch-Nyhan syndrome, severe combined immunodeficiency disease (SCID), gout, renal lithiasis, xanthinuria, orotic aciduria (type I or II), or ornithine transcarbamoylase deficiency.

Non limiting examples of "disorders of steroid metabolism" may include congenital adrenal hyperplasia, 21 -Hydroxylase deficiency or salt-losing congenital adrenal hyperplasia. Non limiting examples of "disorders of mitochondrial function" may include Kearns-Sayre syndrome or external ophthalmoplegia NOS.

Non limiting examples of "disorders of peroxisomal function" may include Zellweger syndrome, X-linked adrenoleukodystrophy, neonatal adrenoleukodystrophy (NALD), rhizomelic chondrodysplasia punctata (RCDP) or infantile Refsum's disease (IRD).

In yet a further embodiment, the invention relates to a method, wherein said liquid solution containing blood sample is analyzed to monitor immune system deficiencies, infectious diseases, allergy, food intolerance, cartilage disorders, inflammation and/or type-1 & type- 2 diabetes.

The term "immune system deficiencies" discloses the immuno-protection state of the patient and includes autoimmunity diseases.

The term "infectious diseases" describes diseases caused by HIV, tetanus, rotavirus and/or adenovirus. "HIV" or "human immunodeficiency virus" is a retrovirus that infects the human immune system and may cause AIDS. "Tetanus" is caused by the bacteria Clostridium tetani which enter the body usually by way of contaminated wounds. Neonatal tetanus is a form of tetanus that occurs in newborn who have not acquired passive immunity because the mother has never been immunized. Human "rotavirus" and "adenovirus" infections are the commonest causes of acute childhood diarrhea, occurring primarily among children less than 2 years of age. Onset is sudden with symptoms of diarrhea and vomiting often with a mild fever.

The term "allergy" describes immunologic sensitivity to an allergen that may occur by inhaling airborne allergens or eating food that contains allergens. The term "allergen" refers to any substance that causes an allergic reaction. The consequence of allergens entering into our body is an immune response wherein a special type of IgE antibodies is produced, causing symptoms and disease in a predisposed person.

"Food tolerance" is the results of a series of complex interactions between ingested food antigens, the digestive tract, tissue mast cells, circulating basophils and food antigen specific IgG. Individuals sensitive for food intolerance must take care to avoid food containing the antigens including but not limited to egg-white, cow's milk, wheat, rye, barley, rice, sesame seed, peanut, soybean, tomato, pork meat, carrot, orange, garlic, cocoa, oc-lactalbumine, β-lactoglobuline, casein, tea and pepper.

The term "cartilage disorders" comprise chondropathy, chondromalacia, polychondritis, osteochondritis dissecan and chondrodysplasia. Cartilage disorders are associated with cartilage metabolic changes, most particularly with abnormalities in type II collagen turnover.

The term "inflammation" describes situations in which myeloperoxidase is released. "Myeloperoxidase" or "MPO" can be used as an index of neutrophil activation and a marker of oxidative stress. Furthermore, MPO has been described as a strong prognostic biomarker in cardiovascular diseases, and its implication has been proved in numerous diseases such as gastrointestinal diseases, cystic fibrosis and chronic obstructive pulmonary disease. Finally, it has been shown that MPO might be involved in rheumatoid arthritis and osteoarthritis.

"Type-1 diabetes" describes diabetes caused by a defect in the immune system which leads to destruction of the insulin-producing cells. "Type-2 diabetes" describes diabetes characterized by insulin deficiency due to other factors than a defect in the immune system.

In an embodiment, the invention relates to a method, wherein a plurality of blood samples from dried blood spots is analyzed automatically. A plurality of blood samples can be analyzed automatically, thus avoiding manual handling and potential contamination of the samples. In an embodiment, a plurality of blood samples is analyzed automatically using an Early Life Automate Platform. The term "Early Life Automate Platform" or "ELAP" describes a complete open automate platform comprising: a dispensing system, a wash system, a spectrophotometer and software for the management of results. The dispensing system includes two independent needles, which are able to aspirate from two adjacent samples, or else, from the same reagent, to be dispensed into two separate wells at once. Each needle is associated to a dilutor with two syringes, one with 2500 μΙ and one with 1000 μΙ, respectively for reagent and sample dispensing. A liquid and clot detector is provided for each needle in order to grant a correct functioning of the system. In an embodiment, the invention provides a method, wherein a plurality of blood samples from dried blood spots is analyzed automatically with an assembly, said assembly comprising a first array and a second array, said method comprising the steps of: providing a plurality of dried blood spots on filter paper; punching a disc of each of said dried blood spot; placing each disc in a first container of said first array; extracting in each first container blood sample from said disc with a liquid solution; inserting said second array in said first array; separating said liquid solution containing blood sample from said disc by removing said liquid solution containing blood sample out of each second container; and analyzing said blood samples in parallel; wherein said assembly permits said liquid solution containing blood sample to be separated from said disc of said dried blood spot.

In an embodiment, the invention provides a method, wherein a plurality of blood samples from dried blood spots is analyzed automatically with an assembly, said assembly comprising a first array and a second array, wherein said first array is incorporated into a microtitre plate and said second array is incorporated into a grid. In a further embodiment, the invention relates to an assembly, wherein said second array is incorporated into a grid with one or more round openings and wherein the diameter of said round openings is smaller than the diameter of said disc of a dried blood spot.

In an embodiment, the invention provides a method, wherein said liquid solution containing said blood sample is removed out of said second container with a needle for automated liquid withdrawal. A needle for automated liquid withdrawal is part of the dispensing unit of the ELAP as described above.

In addition, the present invention relates to a device for analyzing a blood sample from a dried blood spot on a filter paper disc comprising: a first container, adapted to receive at least one filter paper disc, a liquid solution, and a second container, and a second container disposed with one or more openings at or towards the base, adapted for dismountable insertion at least partially into the first container, whereby, said openings fluidly connect the inside of said first container to the inside of said second container when said second container is inserted into said first container, thereby allowing liquid solution from the first container to enter the second container, and said openings are configured to prevent entry of said filter paper disc into said second container.

In an embodiment, the invention relates to a device, wherein said first container is a reaction vessel and said second container is a reaction vessel with at least one opening and wherein the dimension of said opening is smaller than the dimension of said filter paper disc. In a particular embodiment, the invention relates to a device, wherein said second container is a reaction vessel with at least one round opening and wherein the diameter of said round opening is smaller than the diameter of said disc of a dried blood spot. Preferably, the diameter of said round opening may be ranging between 1.0 and 4.8 mm. More preferably, the diameter of said round opening is 1.0, 1 .1 , 1.2, 1 .3, 1 .4, 1.5, 1 .6, 1 .7, 1 .8, 1.9, 2.0, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1 , 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7 or 4.8 mm. Preferably, the diameter of said disc of a dried blood spot may be ranging between 2.0 and 5.0 mm. More preferably, the diameter of said disc is 2.0, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1 , 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or 5.0 mm.

In an embodiment, the invention relates to an assembly comprising: a first array comprising a plurality of first containers as describe above, and a second array comprising a plurality of second containers as described above, wherein the arrays are configured such that said plurality of second containers align with, and are dismountable insertable into said plurality of first containers.

In an embodiment, the invention relates to an assembly, wherein said first array is incorporated into a microtitre plate and said second array is incorporated into a grid. In a particular embodiment, the invention relates to an assembly, wherein said second array is incorporated into a grid with one or more round openings and wherein the diameter of said round openings is smaller than the diameter of said disc of a dried blood spot.

An illustrative but non-limiting example is described hereunder.

EXAMPLES

Example 1 : Neonatal phenylketonuria (PKU) screening assay on ELAP. The neonatal PKU screening assay is an enzymatic assay for the quantitative determination of phenylalanine concentrations in neonates using blood spot samples dried on Whatman S&S 903 filter collection paper. This kit is particularly suitable for use in a neonatal screening program to measure phenylalanine concentrations as an aid in identifying phenylketonuria in newborns. The phenylalanine from the dried blood spot is extracted with trichloroacetic acid as extraction buffer. After extraction, the sample is combined with the enzyme reagent phenylalanine dehydrogenase. This enzyme reagent catalyzes the NAD-dependent oxidative deamination of phenylalanine to phenylpyruvate and ammonia. The produced NADH reacts with a colour reagent in which a tetrazolium salt gets reduced producing a distinct colour. This colour can be measured with a photometer at 550 nm and is directly proportional to the concentration of phenylalanine present in the sample.

The neonatal PKU screening assay on ELAP is advantageous because it obviates the need for additional extraction or filtration steps after extraction of the blood sample from the filter paper disc. The filter paper disc of a dried blood spot is separated from the liquid containing the blood sample, thus allowing efficient and economical analysis of the blood samples. The ELAP procedure comprises the following steps:

- Prepare 10 racked 5 ml extraction tubes (Sarstedt; 55.476.005).

- Punch 8 discs of each 3.2 mm diameter of a dried blood spot and put 8 discs into an extraction tube containing 400 μΙ of trichloroacetic acid (Merck). Ensure that each disc is fully immersed in the liquid.

- Incubate the extraction tubes on an orbital shaker for 30 minutes at 20°C and 900 rpm.

After the incubation, remove the extraction tubes from the shaker and put them on the rack inside the ELAP. The ELAP will perform automated handling of the samples. A first 1 ml sampling tube (Bio-Rad; Titertube Micro Tubes; 223-9390) with two round openings towards the bottom of each 3 mm diameter is placed in a first extraction tube. The positioning of the sampling tube occurs naturally on the bottom of the extraction tube. This permits the liquid to flow freely through the two openings until equal liquid levels are obtained in the extraction tube and the sampling tube.

A needle for automated liquid withdrawal is inserted in the sampling tube and 40 μΙ of the liquid solution containing the blood sample is withdrawn from the sampling tube and transferred to the corresponding well of a microtiter plate.

A second sampling tube is placed in the second extraction tube, 40 μΙ of the liquid solution containing the blood sample is withdrawn from the sampling tube and transferred to the corresponding well of a microtiter plate.

- These steps are repeated until all tubes have been sampled.

- 100 μΙ of the enzyme-coenzyme working solution is added to each well.

The samples are incubated 30 minutes at 20°C, with moderate shaking. 80 μΙ of colour reagent mixture is added to each well.

The optical density is measured at 550 nm after 10 min.

The ELAP software draws a calibration curve. Corresponding phenylalanine concentrations in mg/dl are obtained by interpolating the absorbance of each sample on the calibration curve.

Claims

Method for analyzing a blood sample from a dried blood spot with a device, said device comprising a first container and a second container,

said method comprising the steps of:

a) providing a dried blood spot on a filter paper;

b) punching a disc of said dried blood spot on said filter paper;

c) placing said disc in said first container;

d) extracting in said first container blood sample from said disc with a liquid solution; e) inserting said second container in said first container;

f) separating said liquid solution containing blood sample from said disc by removing said liquid solution containing blood sample out of said second container; and g) analyzing said blood sample;

wherein said device permits said liquid solution containing blood sample to be separated from said disc of said dried blood spot.

Method according to claim 1 , wherein said first container is a reaction vessel and said second container is a reaction vessel with at least one opening and wherein the dimension of said opening is smaller than the dimension of said disc of a dried blood spot.

3. Method according to claim 1 or 2, wherein said liquid solution reaches a pH value ranging from 2.0 to 13.0. 4. Method according to any of claims 1 to 3, wherein said liquid solution containing blood sample provided in step f) contains lysed blood cells.

Method according to any of claims 1 to 4, wherein said liquid solution containing blood sample provided in step f) is analyzed to diagnose neonatal or pediatric disorders.

Method according to any of claims 1 to 5, wherein said liquid solution containing blood sample provided in step f) is analyzed to diagnose inborn errors of metabolism, such as disorders of carbohydrate metabolism, disorders of amino acid and organic acid metabolism, disorders of fatty acid oxidation and mitochondrial metabolism, disorders of porphyrin metabolism, disorders of purine or pyrimidine metabolism, disorders of steroid metabolism, disorders of mitochondrial function, disorders of peroxisomal function.

7. Method according to any of claims 1 to 6, wherein said liquid solution containing blood sample provided in step f) is analyzed to monitor immune system deficiencies, infectious diseases, allergy, food intolerance, cartilage disorders, inflammation and/or type-1 & type-2 diabetes.

8. Method according to any of claims 1 to 7, wherein a plurality of blood samples from dried blood spots on filter paper is analyzed automatically.

9. Method according to claim 8, wherein said liquid solution containing blood sample is removed out of said second container with a needle for automated liquid withdrawal. 10. A device for analyzing a blood sample from a dried blood spot on a filter paper disc comprising:

a first container, adapted to receive at least one filter paper disc, a liquid solution, and a second container, and

a second container disposed with one or more openings, adapted for dismountable insertion at least partially into the first container,

whereby, said openings fluidly connect the inside of said first container to the inside of said second container when said second container is inserted into said first container, and said openings are configured to prevent entry of said filter paper disc into said second container.

1 1 . A device according to claim 10, wherein said first container is a reaction vessel and said second container is a reaction vessel with at least one opening and wherein the dimension of said opening is smaller than the dimension of said filter paper disc. 12. An assembly comprising:

a first array comprising a plurality of first containers according to claim 10, and a second array comprising a plurality of second containers according to claim 10, wherein the arrays are configured such that said plurality of second containers align with, and are dismountable insertable into said plurality of first containers.

13. An assembly according to claim 12, wherein said first array is incorporated into a microtitre plate and said second array is incorporated into a grid.