WO2010043668A1 - Dried blood spots for blood analysis - Google Patents
Dried blood spots for blood analysis Download PDFInfo
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- WO2010043668A1 WO2010043668A1 PCT/EP2009/063470 EP2009063470W WO2010043668A1 WO 2010043668 A1 WO2010043668 A1 WO 2010043668A1 EP 2009063470 W EP2009063470 W EP 2009063470W WO 2010043668 A1 WO2010043668 A1 WO 2010043668A1
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- WO
- WIPO (PCT)
- Prior art keywords
- filter paper
- blood
- cellulosic filter
- blood sample
- degradable
- Prior art date
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
- G01N2001/2873—Cutting or cleaving
- G01N2001/288—Filter punches
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/483—Physical analysis of biological material
- G01N33/487—Physical analysis of biological material of liquid biological material
- G01N33/49—Blood
Definitions
- the present invention relates to a method for analyzing blood samples from dried blood spots on filter paper.
- the invention also relates to the use of cellulase for the digestion or degradation of the cellulosic filter paper.
- Dried blood spot or DBS method consists of blotting few drops of blood onto a paper especially designed to absorb blood drops for laboratory analysis. Blood drops are obtained thanks to a light incision carried out to the heel or the ear lobe.
- This method is slightly invasive and generally painless. This is the standard procedure when dealing with newborn diagnosis in paediatrics, prenatology or neonatology.
- the use of paper cards, such as Guthrie card, for the collection of blood sample is particularly suitable for the storage and transport of blood samples at the time of epidemiologic studies or of screening of population. These cards are usually filter paper made of cotton linters. Other well-known advantages of this method are the ease of collection, the low volume of storage, and the transport without need for refrigeration.
- 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 disk is punched out from the spot and loaded into a multiwell plate, where it is extracted with various solvents.
- a sample might first be immersed for several hours in a methanol solution.
- the sample is analyzed by standard spectroscopic methods, e.g. mass spectroscopy, high performance liquid chromatography or gas chromatography. The extraction is necessary since the paper piece which has been used for blood collection is still present in the multiwell plate.
- the extraction step causes several drawbacks.
- the degradation of the filter paper has been studied.
- US 5,432,097 discloses the use of specific enzyme to digest the filter paper.
- the degradation of the filter paper is partial and the supernatant have to be extracted at least four times from the compacted paper underlayer. Therefore, there is a need for the analysis of dried blood spots without resort to filtration or extraction step. There exits the need to circumvent the presence of the paper card and to allow efficient and fast testing of blood samples.
- the inventors realised that providing a method comprising the entire degradation of the cellulosic filter paper can greatly improve the efficiency of the standard procedure.
- the present invention relates to a method for analyzing blood sample from dried blood spots on filter paper comprising the steps of: a) providing a degradable cellulosic filter paper, b) placing a blood sample on said degradable cellulosic filter paper, c) drying said blood sample on said degradable cellulosic filter paper, thus providing a dried blood spot, d) punching out a small disc of said dried blood spot and moving it to a reaction vessel, e) degrading said degradable cellulosic filter paper from said dried blood spot with a cellulase in presence of an aqueous acidic solution, thus providing a blood sample; and, f) analyzing said blood sample, wherein said cellulase is selected from the group comprising endoglucanase, exoglucanase, hemicellulase and/or beta-glucosidase.
- said agitation of step e) reaches a speed value ranging from 300 to 900rpm.
- the use of a combination of enzymes can dramatically improve the degradation of the cellulosic filter paper from the dried blood spot. Moreover, the degradation of the cellulosic filter paper does not alter the blood sample which can be normally used afterwards.
- the complete digestion of the cellulosic filter paper avoids a filtration step in the process of blood sample analysis.
- the dried blood spot can be automatically process in deep-well plates or similar reaction vessel, thus avoiding manual handling and potential contamination of the sample.
- the method of the present invention improves the time needed for the analysis. Therefore, more samples can be handled and analyzed per hour or per day.
- the degradation of the cellulosic filter paper can greatly be improved by a specific combination of an enzymatic process and a physical or chemical process.
- the method may combine an enzymatic process and an ultrasound treatment.
- Cellulase or Cellulytic enzymes are enzymes involved in hydrolyse of cellulose.
- the term "1 ,4-beta-D-glucan cellobiohydrase or exoglucanase or cellobiohydrolase” refers to an enzyme of general class E. C. 3.2.1.91 (Enzyme Classification), which is able to perform the hydrolysis of cellulose end chain, thus releasing cellobiose units.
- the term "endo-beta-1 ,4-glucanase, endoglucanase or endo-1 ,4-beta-D- glucan 4-glucanohydrolase” refers to an enzyme of general class E. C. 3.2.1.4 (Enzyme Classification), which is able to perform the endohydrolysis of (1 ⁇ 4)- ⁇ -D-glucosidic linkages in cellulose, lichenin and cereal ⁇ -D-glucans.
- beta-glucosidase refers to an enzyme of general class E. C. 3.2.1.21 (Enzyme Classification), which is able to hydrolyse the terminal, non-reducing ⁇ - D-glucose residues with release of ⁇ -D-glucose.
- degradation or digestion refers to break down polymeric macromolecules into their smaller building blocks.
- the present invention relates to a method for analyzing blood sample from dried blood spot on filter paper comprising the steps of: a) providing a degradable cellulosic filter paper, b) placing a blood sample on said degradable cellulosic filter paper, c) drying said blood sample on said degradable cellulosic filter paper, thus providing a dried blood spot, d) punching out a small disc of said dried blood spot and moving it to a reaction vessel, e) degrading said degradable cellulosic filter paper from said dried blood spot with a cellulase in presence of an aqueous acidic solution and agitating said solution, thus providing a blood sample; and f) analyzing said blood sample; wherein said cellulase is selected from the group comprising endoglucanase, exoglucanase, hemicellulase and/or beta-glucosidase.
- said aqueous acidic solution may reach a pH value ranging from 2.0 to 8.0.
- the pH value of said aqueous acidic solution may range from 3.0 to 7.0, more preferably from 4.0 to 6.0.
- the pH value of said aqueous acidic solution may be 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1 , 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 or 6.0, or a value in the range between any two of the aforementioned values.
- the pH value may allow to adapt the activity of the cellulase enzyme and thus to reduce the process time to a minimum.
- the agitation of the solution in step e) is preferably done at a speed value ranging from 300 to 900rpm and can be done using conventional shakers or agitators known in the art, capable of agitating a fluid containing container, reservoir, recipient, tube, EppendorfTM tube or bottle.
- said aqueous acidic solution may have a concentration of at least 0.01 molar.
- said degradable cellulosic filter paper may be degraded in an aqueous acidic solution of at least 0.1 molar.
- said degradable cellulosic filter paper may be degraded in an aqueous acidic solution having a concentration ranging from 0.1 molar to 5 molar. More preferably, said degradable cellulosic filter paper may be degraded in an aqueous acidic solution having a concentration ranging from 0.1 molar to 3 molar.
- said degradable cellulosic filter paper may be degraded in an aqueous acidic solution having a concentration of 0.1 , 0.5, 1.0, 1.5, 2.0, 2.5 or 3.0 molar, or a value in the range between any two of the aforementioned values.
- said aqueous acidic solution may contain hydrochloric acid, hydrofluoric acid, perchloric acid, thiocyanic acid, nitrous acid, chromic acid, methanesulfonic acid, trifluoromethanesulfonic acid, sulphuric acid, nitric acid, acetic acid, phosphate buffer, phosphoric acid, bromic acid, hypochloric acid, sulfinic acid, sulfonic acid, fumaric acid or carboxylic acids of general formula R-C(O)OH wherein R is CH 3 , CH 2 NO 2 , CH 2 F, CH 2 CI, CH 2 Br, CH 2 I, CHCI 2 , CCI 3 , CF 3 , H, HO, C 6 H 5 , 0-O 2 NC 6 H 4 , p- O 2 NC 6 H 4 , m-O 2 NC 6 H 4 , 0-ClC 6 H 4 , P-CIC 6 H 4 , m-CIC 6
- said aqueous acidic solution may contain hydrochloric acid, sulphuric acid, nitric acid, acetic acid, phosphate buffer, phosphoric acid, bromic acid, hypochloric acid, sulfinic acid, sulfonic acid, fumaric acid. More preferably, said aqueous acidic solution may contain hydrochloric acid, acetic acid or phosphate buffer.
- said degradable cellulosic filter paper may be degraded in an aqueous acidic solution of at least 0.1 molar containing hydrochloric acid, acetic acid or phosphate buffer.
- said degradable cellulosic filter paper may be degraded in an aqueous acidic solution having a concentration ranging from 0.1 molar to 5 molar and containing hydrochloric acid, acetic acid or phosphate buffer.
- said degradable cellulosic filter paper may be degraded in an aqueous acidic solution having a concentration ranging from 0.1 molar to 3 molar and containing hydrochloric acid, acetic acid or phosphate buffer.
- said degradable cellulosic filter paper may be degraded in an aqueous acidic solution having a concentration of 0.1 , 0.5, 1.0, 1.5, 2.0, 2.5 or 3.0 molar, or a value in the range between any two of the aforementioned values, and containing hydrochloric acid, acetic acid or phosphate buffer.
- the degradation of said degradable cellulosic filter paper performed in step e) may occur at a temperature ranging from 30 0 C to 70 0 C.
- the activity of the cellulase enzyme can be also adapted by modulating the temperature.
- the degradation of said degradable cellulosic filter paper of step e) may occur at a temperature ranging from 37°C to 65°C, more preferably from 37°C to 60 0 C.
- the degradation of said degradable cellulosic filter paper of step e) may occur at a temperature of 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59 or 60 0 C, or a value in the range between any two of the aforementioned values.
- the degradation of said degradable cellulosic filter paper may be performed for 1 to 120 minutes.
- said degradation of said degradable cellulosic filter paper may be performed for 1 to 90 minutes, more preferably for 1 to 60 minutes.
- said degradation of said degradable cellulosic filter paper 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.
- cellulases may include cellulases from Penicillium, Trichoderma, Humicola, Fusarium, Thermomonospora, Cellulomonas, Clostridium and Aspergillus.
- cellulases may be produced from Trichoderma fungi.
- Trichoderma include Trichoderma reesei, Trichoderma longibrachiatum, Trichoderma viride, Trichoderma koningii.
- said cellulase may be produced with a genetically modified fungus from Trichoderma reesei.
- said degradable cellulosic filter paper from said dried blood spot may be degraded with a combination of exoglucanase, endoglucanase and beta- glucosidase enzymes.
- said degradable cellulosic filter paper from said dried blood spot may be degraded with a cellulase sold under the trademark AccelleraseTM 1000 and supplied by Genencor.
- the method may further comprise the step of submitting said degradable cellulosic filter paper to a physical or chemical treatment.
- the method may further comprise the step of submitting said degradable cellulosic filter paper to a physical treatment.
- This combination of an enzymatic and a chemical/physical process improve the degradation of the filter paper and reduce the time required for the analysis.
- the term "physical treatment" may refer to the exposure of said cellulosic filter paper to ultrasound, magnetic field, ultraviolet, infrared, microwave, electrical field, freezing or heating.
- the term "chemical treatment” may refer to contacting the degradable cellulosic filter paper with salts, detergent or oxidant (e.g. sodium hypochlorite, hydrogen peroxide).
- salts e.g. sodium hypochlorite, hydrogen peroxide.
- the method may further comprise the step of submitting said degradable cellulosic filter paper to an ultrasound treatment prior to step e) or simultaneous to step e). In another preferred embodiment, the method may further comprise the step of submitting said degradable cellulosic filter paper to an ultrasound treatment prior to step e). Alternatively, the method may further comprise the step of submitting said degradable cellulosic filter paper to an ultrasound treatment simultaneous to step e).
- the combination of ultrasound with a cellulase can avoid the use of non- environmental friendly solvent or components.
- Said ultrasound treatment may consist of submitting said degradable cellulosic filter paper to an ultrasound field at frequencies in the range of 1 to 1000 kHz.
- said degradable cellulosic filter paper may be submitted to an ultrasound field at frequencies ranging from 10 to 500 kHz, more preferably from 50 to 250 kHz.
- said degradable cellulosic filter paper may be submitted to an ultrasound field at frequencies of 50, 60, 70, 80, 90, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240 or 250 kHz, or a value in the range between any two of the aforementioned values.
- said degradable cellulosic filter paper may be submitted to an ultrasound field for a reaction time ranging from 1 second to 5 minutes.
- said degradable cellulosic filter paper may be submitted to an ultrasound field for a reaction time ranging from 1 second to 1 minute, more preferably from 1 second to 45 seconds.
- said degradable cellulosic filter paper may be submitted to an ultrasound field for a reaction time of 1 , 5, 10, 15, 20, 25, 30, 35, 40 or 45 seconds, or a value in the range between any two of the aforementioned values.
- said method may further comprise the step of adding to the reaction vessel magnetic beads and submitting said reaction vessel to a magnetic stirrer.
- said blood sample provided in step e) may be analyzed to detect endocrine disorders, e.g. congenital hypothyroidism (TSH), congenital adrenal hyperplasia.
- said blood sample provided in step e) may be analyzed to detect blood cell disorders such as glucose-6-phosphate dehydrogenase deficiency.
- said blood sample provided in step e) may be analyzed to detect 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.
- 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, tyrosinemia (type I, Il or III), alcaptonuria, homocystinuria, histidinemia, maple syrup urine disease (MSUD type Ib 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), heartnup disease or hyperammonemia-hyperornithinemia-homocitrullinuroa syndrome.
- phenylketonuria type
- 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 Il (CPT II) deficiency.
- VLCAD very-long-chain acyl-CoA dehydrogenase deficiency
- LCAD long-chain acyl-CoA dehydrogenase deficiency
- MCAD medium-chain acyl-CoA dehydrogenase defici
- 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).
- CEP congenital erythropoietic porphyria
- EPP erythropoietic protoporphyria
- ADP ALA dehydratase deficiency porphyria
- HCP hereditary coproporphyria
- VP variegate porphyria
- PCT hepatoerythropoietic porphyria
- 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).
- said blood sample provided in step e) may be analyzed to detect phenylketonuria.
- said blood sample of step f) may be analyzed to detect maple syrup urine disease.
- said blood sample of step f) may be analyzed to detect phenylketonuria and maple syrup urine disease.
- said blood sample provided in step e) may contain lysed blood cells.
- Lysed blood cells refer to blood cells having their cellular membrane deteriorated or broken.
- the present invention is particularly useful for the degradation of cellulosic filter paper.
- the use of a combination of endoglucanase, exoglucanase, hemicellulase and/or beta-glucosidase enzymes and/or ultrasound treatment is suitable for the degradation of cellulosic filter paper.
- the use of endoglucanase, exoglucanase, beta-glucosidase enzymes and ultrasound treatment is particularly suitable for the degradation of cellulosic filter paper.
- the present invention relates to a method for the analysis in parallel of a plurality of blood samples from dried blood spots comprising the steps of: a) providing a plurality of dried blood spots, b) punching out a small disc of said dried blood spots and moving each disc to a different well of a multiwell plate, c) degrading the dried blood spots with a cellulase in presence of an aqueous acidic solution, thus providing a plurality of blood samples, and d) analyzing said blood sample in parallel,
- cellulase is selected from the group comprising endoglucanase, exoglucanase, hemicellulase and/or beta-glucosidase
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Abstract
The present invention relates to a method for analysing blood samples from dried blood spots on filter paper. In particular, the method of the invention may comprise the steps of: a) providing a degradable cellulosic filter paper, b) placing a blood sample on said degradable cellulosic filter paper, c) drying said blood sample on said degradable cellulosic filter paper, thus providing a dried blood spot, d) punching out a small disc of said dried blood spot and moving it to a reaction vessel, e) degrading said degradable cellulosic filter paper from said dried blood spot with a cellulase in presence of an aqueous acidic solution, thus providing a blood sample, and f) analyzing said blood sample; wherein said cellulase is selected from the group comprising endoglucanase, exoglucanase, hemicellulase and/or beta-glucosidase.
Description
DRIED BLOOD SPOTS FOR BLOOD ANALYSIS
FIELD OF THE INVENTION
The present invention relates to a method for analyzing blood samples from dried blood spots on filter paper. The invention also relates to the use of cellulase for the digestion or degradation of the cellulosic filter paper.
BACKGROUND OF THE INVENTION
Dried blood spot or DBS method consists of blotting few drops of blood onto a paper especially designed to absorb blood drops for laboratory analysis. Blood drops are obtained thanks to a light incision carried out to the heel or the ear lobe. This method is slightly invasive and generally painless. This is the standard procedure when dealing with newborn diagnosis in paediatrics, prenatology or neonatology. The use of paper cards, such as Guthrie card, for the collection of blood sample is particularly suitable for the storage and transport of blood samples at the time of epidemiologic studies or of screening of population. These cards are usually filter paper made of cotton linters. Other well-known advantages of this method are the ease of collection, the low volume of storage, and the transport without need for refrigeration.
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 disk is punched out from the spot and loaded into a multiwell plate, where it is extracted with various solvents. For example, a sample might first be immersed for several hours in a methanol solution. After an extraction step, the sample is analyzed by standard spectroscopic methods, e.g. mass spectroscopy, high performance liquid chromatography or gas chromatography. The extraction is necessary since the paper piece which has been used for blood collection is still present in the multiwell plate.
The extraction step causes several drawbacks. The degradation of the filter paper has been studied. For example, US 5,432,097 discloses the use of specific enzyme to digest the filter paper. However, the degradation of the filter paper is partial and the supernatant have to be extracted at least four times from the compacted paper underlayer.
Therefore, there is a need for the analysis of dried blood spots without resort to filtration or extraction step. There exits the need to circumvent the presence of the paper card and to allow efficient and fast testing of blood samples.
SUMMARY OF THE INVENTION
The aspects of the present invention address at least some, e.g., one or more, of the above discussed needs of the art.
In particular, the inventors realised that providing a method comprising the entire degradation of the cellulosic filter paper can greatly improve the efficiency of the standard procedure.
The present invention relates to a method for analyzing blood sample from dried blood spots on filter paper comprising the steps of: a) providing a degradable cellulosic filter paper, b) placing a blood sample on said degradable cellulosic filter paper, c) drying said blood sample on said degradable cellulosic filter paper, thus providing a dried blood spot, d) punching out a small disc of said dried blood spot and moving it to a reaction vessel, e) degrading said degradable cellulosic filter paper from said dried blood spot with a cellulase in presence of an aqueous acidic solution, thus providing a blood sample; and, f) analyzing said blood sample, wherein said cellulase is selected from the group comprising endoglucanase, exoglucanase, hemicellulase and/or beta-glucosidase.
Preferably, said agitation of step e) reaches a speed value ranging from 300 to 900rpm.
The use of a combination of enzymes can dramatically improve the degradation of the cellulosic filter paper from the dried blood spot. Moreover, the degradation of the cellulosic filter paper does not alter the blood sample which can be normally used afterwards. The complete digestion of the cellulosic filter paper avoids a filtration step in the process of blood sample analysis. The dried blood spot can be automatically process in deep-well plates or similar reaction vessel, thus avoiding manual handling and potential contamination of the sample. The method of the present invention improves the time
needed for the analysis. Therefore, more samples can be handled and analyzed per hour or per day.
More specifically, the degradation of the cellulosic filter paper can greatly be improved by a specific combination of an enzymatic process and a physical or chemical process. In particular, the method may combine an enzymatic process and an ultrasound treatment.
Related aspects concern the use of enzymatic and physical process for the degradation of cellulosic filter paper. These aspects and preferred embodiments of the invention are described in the following sections and in the appended claims. Others advantages of the present invention will become clear from the following more detailed description and the following examples.
DETAILED DESCRIPTION Cellulase or Cellulytic enzymes are enzymes involved in hydrolyse of cellulose.
As used herein, the term "1 ,4-beta-D-glucan cellobiohydrase or exoglucanase or cellobiohydrolase" refers to an enzyme of general class E. C. 3.2.1.91 (Enzyme Classification), which is able to perform the hydrolysis of cellulose end chain, thus releasing cellobiose units. As used herein, the term "endo-beta-1 ,4-glucanase, endoglucanase or endo-1 ,4-beta-D- glucan 4-glucanohydrolase" refers to an enzyme of general class E. C. 3.2.1.4 (Enzyme Classification), which is able to perform the endohydrolysis of (1 →4)-β-D-glucosidic linkages in cellulose, lichenin and cereal β-D-glucans.
As used herein, the term "beta-glucosidase" refers to an enzyme of general class E. C. 3.2.1.21 (Enzyme Classification), which is able to hydrolyse the terminal, non-reducing β- D-glucose residues with release of β-D-glucose.
As used herein the term "degradation or digestion" refers to break down polymeric macromolecules into their smaller building blocks.
The present invention relates to a method for analyzing blood sample from dried blood spot on filter paper comprising the steps of: a) providing a degradable cellulosic filter paper, b) placing a blood sample on said degradable cellulosic filter paper,
c) drying said blood sample on said degradable cellulosic filter paper, thus providing a dried blood spot, d) punching out a small disc of said dried blood spot and moving it to a reaction vessel, e) degrading said degradable cellulosic filter paper from said dried blood spot with a cellulase in presence of an aqueous acidic solution and agitating said solution, thus providing a blood sample; and f) analyzing said blood sample; wherein said cellulase is selected from the group comprising endoglucanase, exoglucanase, hemicellulase and/or beta-glucosidase.
In one embodiment of the present invention, said aqueous acidic solution may reach a pH value ranging from 2.0 to 8.0. Preferably, the pH value of said aqueous acidic solution may range from 3.0 to 7.0, more preferably from 4.0 to 6.0. In a preferred embodiment, the pH value of said aqueous acidic solution may be 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1 , 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 or 6.0, or a value in the range between any two of the aforementioned values. The pH value may allow to adapt the activity of the cellulase enzyme and thus to reduce the process time to a minimum.
The agitation of the solution in step e) is preferably done at a speed value ranging from 300 to 900rpm and can be done using conventional shakers or agitators known in the art, capable of agitating a fluid containing container, reservoir, recipient, tube, Eppendorf™ tube or bottle.
In another embodiment, said aqueous acidic solution may have a concentration of at least 0.01 molar. Preferably, said degradable cellulosic filter paper may be degraded in an aqueous acidic solution of at least 0.1 molar. Preferably, said degradable cellulosic filter paper may be degraded in an aqueous acidic solution having a concentration ranging from 0.1 molar to 5 molar. More preferably, said degradable cellulosic filter paper may be degraded in an aqueous acidic solution having a concentration ranging from 0.1 molar to 3 molar. In particular, said degradable cellulosic filter paper may be degraded in an aqueous acidic solution having a concentration of 0.1 , 0.5, 1.0, 1.5, 2.0, 2.5 or 3.0 molar, or a value in the range between any two of the aforementioned values.
In another embodiment, said aqueous acidic solution may contain hydrochloric acid, hydrofluoric acid, perchloric acid, thiocyanic acid, nitrous acid, chromic acid,
methanesulfonic acid, trifluoromethanesulfonic acid, sulphuric acid, nitric acid, acetic acid, phosphate buffer, phosphoric acid, bromic acid, hypochloric acid, sulfinic acid, sulfonic acid, fumaric acid or carboxylic acids of general formula R-C(O)OH wherein R is CH3, CH2NO2, CH2F, CH2CI, CH2Br, CH2I, CHCI2, CCI3, CF3, H, HO, C6H5, 0-O2NC6H4, p- O2NC6H4, m-O2NC6H4, 0-ClC6H4, P-CIC6H4, m-CIC6H4, o-(CH3)3N+C6H4, p-(CH3)3N+C6H4 or P-OMeC6H4.
In a preferred embodiment, said aqueous acidic solution may contain hydrochloric acid, sulphuric acid, nitric acid, acetic acid, phosphate buffer, phosphoric acid, bromic acid, hypochloric acid, sulfinic acid, sulfonic acid, fumaric acid. More preferably, said aqueous acidic solution may contain hydrochloric acid, acetic acid or phosphate buffer.
In another embodiment, said degradable cellulosic filter paper may be degraded in an aqueous acidic solution of at least 0.1 molar containing hydrochloric acid, acetic acid or phosphate buffer. In a preferred embodiment, said degradable cellulosic filter paper may be degraded in an aqueous acidic solution having a concentration ranging from 0.1 molar to 5 molar and containing hydrochloric acid, acetic acid or phosphate buffer. Preferably, said degradable cellulosic filter paper may be degraded in an aqueous acidic solution having a concentration ranging from 0.1 molar to 3 molar and containing hydrochloric acid, acetic acid or phosphate buffer. In particular, said degradable cellulosic filter paper may be degraded in an aqueous acidic solution having a concentration of 0.1 , 0.5, 1.0, 1.5, 2.0, 2.5 or 3.0 molar, or a value in the range between any two of the aforementioned values, and containing hydrochloric acid, acetic acid or phosphate buffer.
In another embodiment, the degradation of said degradable cellulosic filter paper performed in step e) may occur at a temperature ranging from 300C to 700C. The activity of the cellulase enzyme can be also adapted by modulating the temperature. Preferably, the degradation of said degradable cellulosic filter paper of step e) may occur at a temperature ranging from 37°C to 65°C, more preferably from 37°C to 600C. In a preferred embodiment, the degradation of said degradable cellulosic filter paper of step e) may occur at a temperature of 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59 or 600C, or a value in the range between any two of the aforementioned values.
In another embodiment, the degradation of said degradable cellulosic filter paper (step e)) may be performed for 1 to 120 minutes. Preferably, said degradation of said degradable
cellulosic filter paper may be performed for 1 to 90 minutes, more preferably for 1 to 60 minutes. In a preferred embodiment, said degradation of said degradable cellulosic filter paper 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.
Examples of cellulases may include cellulases from Penicillium, Trichoderma, Humicola, Fusarium, Thermomonospora, Cellulomonas, Clostridium and Aspergillus. Preferably, cellulases may be produced from Trichoderma fungi. Trichoderma include Trichoderma reesei, Trichoderma longibrachiatum, Trichoderma viride, Trichoderma koningii. In a preferred embodiment, said cellulase may be produced with a genetically modified fungus from Trichoderma reesei.
In a preferred embodiment, said degradable cellulosic filter paper from said dried blood spot may be degraded with a combination of exoglucanase, endoglucanase and beta- glucosidase enzymes. In a preferred embodiment, said degradable cellulosic filter paper from said dried blood spot may be degraded with a cellulase sold under the trademark Accellerase™ 1000 and supplied by Genencor.
In another preferred embodiment, the method may further comprise the step of submitting said degradable cellulosic filter paper to a physical or chemical treatment. Preferably, the method may further comprise the step of submitting said degradable cellulosic filter paper to a physical treatment. This combination of an enzymatic and a chemical/physical process improve the degradation of the filter paper and reduce the time required for the analysis. As used herein, the term "physical treatment" may refer to the exposure of said cellulosic filter paper to ultrasound, magnetic field, ultraviolet, infrared, microwave, electrical field, freezing or heating.
As used herein, the term "chemical treatment" may refer to contacting the degradable cellulosic filter paper with salts, detergent or oxidant (e.g. sodium hypochlorite, hydrogen peroxide).
In another preferred embodiment, the method may further comprise the step of submitting said degradable cellulosic filter paper to an ultrasound treatment prior to step e) or simultaneous to step e). In another preferred embodiment, the method may further comprise the step of submitting said degradable cellulosic filter paper to an ultrasound
treatment prior to step e). Alternatively, the method may further comprise the step of submitting said degradable cellulosic filter paper to an ultrasound treatment simultaneous to step e). The combination of ultrasound with a cellulase can avoid the use of non- environmental friendly solvent or components.
Said ultrasound treatment may consist of submitting said degradable cellulosic filter paper to an ultrasound field at frequencies in the range of 1 to 1000 kHz. Preferably, said degradable cellulosic filter paper may be submitted to an ultrasound field at frequencies ranging from 10 to 500 kHz, more preferably from 50 to 250 kHz. Preferably, said degradable cellulosic filter paper may be submitted to an ultrasound field at frequencies of 50, 60, 70, 80, 90, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240 or 250 kHz, or a value in the range between any two of the aforementioned values.
In another preferred embodiment, said degradable cellulosic filter paper may be submitted to an ultrasound field for a reaction time ranging from 1 second to 5 minutes. Preferably, said degradable cellulosic filter paper may be submitted to an ultrasound field for a reaction time ranging from 1 second to 1 minute, more preferably from 1 second to 45 seconds. In particular, said degradable cellulosic filter paper may be submitted to an ultrasound field for a reaction time of 1 , 5, 10, 15, 20, 25, 30, 35, 40 or 45 seconds, or a value in the range between any two of the aforementioned values.
In a preferred embodiment, said method may further comprise the step of adding to the reaction vessel magnetic beads and submitting said reaction vessel to a magnetic stirrer.
In another embodiment, said blood sample provided in step e) may be analyzed to detect endocrine disorders, e.g. congenital hypothyroidism (TSH), congenital adrenal hyperplasia. In another embodiment, said blood sample provided in step e) may be analyzed to detect blood cell disorders such as glucose-6-phosphate dehydrogenase deficiency.
In another embodiment, said blood sample provided in step e) may be analyzed to detect 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, tyrosinemia (type I, Il or III), alcaptonuria, homocystinuria, histidinemia, maple syrup urine disease (MSUD type Ib 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 Il (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 a preferred embodiment, said blood sample provided in step e) may be analyzed to detect phenylketonuria. In a preferred embodiment, said blood sample of step f) may be analyzed to detect maple syrup urine disease. Alternatively, said blood sample of step f) may be analyzed to detect phenylketonuria and maple syrup urine disease.
In a preferred embodiment, said blood sample provided in step e) may contain lysed blood cells. Lysed blood cells refer to blood cells having their cellular membrane deteriorated or broken.
The present invention is particularly useful for the degradation of cellulosic filter paper. In particular, the use of a combination of endoglucanase, exoglucanase, hemicellulase and/or beta-glucosidase enzymes and/or ultrasound treatment is suitable for the degradation of cellulosic filter paper. In a preferred embodiment, the use of endoglucanase, exoglucanase, beta-glucosidase enzymes and ultrasound treatment is particularly suitable for the degradation of cellulosic filter paper.
The dried blood spot can be automatically process in deep-well plates or similar reaction vessel, thus avoiding manual handling and potential contamination of the sample. Therefore, in another aspect, the present invention relates to a method for the analysis in parallel of a plurality of blood samples from dried blood spots comprising the steps of: a) providing a plurality of dried blood spots, b) punching out a small disc of said dried blood spots and moving each disc to a different well of a multiwell plate, c) degrading the dried blood spots with a cellulase in presence of an aqueous acidic solution, thus providing a plurality of blood samples, and d) analyzing said blood sample in parallel,
wherein said cellulase is selected from the group comprising endoglucanase, exoglucanase, hemicellulase and/or beta-glucosidase
Claims
1 . A method for analyzing blood sample from dried blood spot on filter paper comprising the steps of: a) providing a degradable cellulosic filter paper, b) placing a blood sample on said degradable cellulosic filter paper, c) drying said blood sample on said degradable cellulosic filter paper, thus providing a dried blood spot, d) punching out a small disc of said dried blood spot and moving it to a reaction vessel, e) degrading said degradable cellulosic filter paper from said dried blood spot with a cellulase in presence of an aqueous acidic solution, and agitating said solution, thus providing a blood sample, and f) analyzing said blood sample; g) wherein said cellulase is selected from the group comprising endoglucanase, exoglucanase, hemicellulase and/or beta-glucosidase.
2. The method according to claim 1 , wherein said agitation of step e) reaches a speed value ranging from 300 to 900rpm.
3. The method according to claim 1 or 2, wherein said aqueous acidic solution of step e) reaches a pH value ranging from 3.0 to 7.0.
4. The method according to any one of claims 1 to 3, wherein the degradation step e) occurs at a temperature ranging from 37°C to 600C.
5. The method according to any one of claims 1 to 4, wherein degradation of said degradable cellulosic filter paper of step e) is performed for 1 to 90 minutes.
6. The method according to any one of claims 1 to 5, wherein said cellulase is produced with a genetically modified fungus from Trichoderma reesei.
7. The method according to any one of claims 1 to 6, wherein said degradable cellulosic filter paper from said dried blood spot is degraded with a combination of exoglucanase, endoglucanase and beta-glucosidase enzymes.
8. The method according to any one of claims 1 to 7, further comprising the step of submitting said degradable cellulosic filter paper to an ultrasound treatment prior to step e) or simultaneous to step e).
9. The method according to claim 8, wherein said ultrasound treatment comprises submitting said degradable cellulosic filter paper to an ultrasound field at frequencies in the range of 1 to 1000 kHz, preferably for at least 1 second to up to 5 minutes.
10. The method according to any one of claims 1 to 9, which further comprises the step of adding to the reaction vessel magnetic beads and submitting said reaction vessel to a magnetic stirrer.
1 1. The method according to any one of claims 1 to 10, wherein said degradable cellulosic filter paper is degraded in step e) in an aqueous acidic solution of at least 0.1 molar containing hydrochloric acid, acetic acid or phosphate buffer.
12. The method according to any one of claims 1 to 1 1 , wherein said blood sample provided in step e) is analyzed to detect phenylketonuria and maple syrup urine disease.
13. The method according to any one of claims 1 to 12, wherein said blood sample provided in step e) contains lysed blood cells.
14. Use of a combination of endoglucanase, exoglucanase, hemicellulase and/or beta- glucosidase enzymes and/or sonication treatment for the degradation of cellulosic filter paper.
15. A method for the analysis in parallel of a plurality of blood samples from dried blood spots comprising the steps of: a) providing a plurality of dried blood spots, b) punching out a small disc of said dried blood spots and moving each disc to a different well of a multiwell plate, c) degrading the dried blood spots with a cellulase in presence of an aqueous acidic solution, thus providing a plurality of blood samples, and d) analyzing said blood sample in parallel, wherein said cellulase is selected from the group comprising endoglucanase, exoglucanase, hemicellulase and/or beta-glucosidase.
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| EP08166899 | 2008-10-17 | ||
| EP08166899.8 | 2008-10-17 |
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