WO2008030154A1 - Agent d'hémolyse - Google Patents

Agent d'hémolyse Download PDF

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Publication number
WO2008030154A1
WO2008030154A1 PCT/SE2007/000747 SE2007000747W WO2008030154A1 WO 2008030154 A1 WO2008030154 A1 WO 2008030154A1 SE 2007000747 W SE2007000747 W SE 2007000747W WO 2008030154 A1 WO2008030154 A1 WO 2008030154A1
Authority
WO
WIPO (PCT)
Prior art keywords
haemolysing
cavity
purified
agent
haemolysing agent
Prior art date
Application number
PCT/SE2007/000747
Other languages
English (en)
Inventor
Bertil Johnny Ingemar Svensson
Lena Margaretha Svensson
Original Assignee
Hemocue Ab
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from SE0601852A external-priority patent/SE0601852L/xx
Application filed by Hemocue Ab filed Critical Hemocue Ab
Publication of WO2008030154A1 publication Critical patent/WO2008030154A1/fr

Links

Classifications

    • 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
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
    • G01N33/721—Haemoglobin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/415—Assays involving biological materials from specific organisms or of a specific nature from plants

Definitions

  • the present invention concerns a device for analysis of blood samples. Specifically the invention concerns a device including a dried and purified Quillaja saponin as haemolysing agent.
  • Devices useful for analysis of components in blood samples are known from e.g. the U.S. Pat. No. 5,674,457.
  • the devices according to this patent are currently widely used for haemoglobin analysis of undiluted whole blood.
  • the devices contain dried reagents, haemolysing agent(s) and optionally other additives adapted to the intended analysis.
  • An initial step of the analysis procedure is the lysis of red blood cells, the presence of which could otherwise disturb the determination of the component to be analysed.
  • haemolysing agents In order to achieve the haemolysis, a wide variety of haemolysing agents have been proposed but only a few such agents have appeared to be useful in practice.
  • Saponins are a group of plant glycosides. They exist in many forms and in many different plants, such as in the roots of Primula veris (cowslip) and Saponaria officinalis, and in the bark of Quillaja saponaria. The saponins have different properties depending on their origin. The properties of different saponin materials also depend on the degree and type of purification. Saponins are widely investigated and used as e.g. adjuvant in vaccines and as detergents in shampoos.
  • White Saponin One saponin haemolysing agent used in practice in the known devices is so-called White Saponin, which has proven to be useful for the determination of glucose as is disclosed in the US patent 5,278,047.
  • a water solution including the saponin and other reagents necessary for the determination are introduced into the device, which is subsequently subjected to a drying procedure.
  • it is necessary to perform the drying by lyophilization which is an elaborate and time consuming method of drying, requiring large and expensive cooling and vacuum equipment.
  • haemolysing agent a solution of which can be dried, within an analysis device, in a less elaborate way.
  • the haemolysing agent should have a high selectivity for red blood cells, i.e. should affect other cells or components, in the blood sample to be analysed, to a lesser extent than the red blood cells, preferably the affect should be negligible.
  • haemolysing agents which haemolyse not only efficiently but also rapidly.
  • a haemolysing agent originating from Quillaja plants eliminates or reduces the problems with haemolysing agents currently proposed and used in devices for analysis of blood samples.
  • the inventive haemolysing agent is a highly purified form of a Quillaja saponin.
  • the inventive Quillaja haemolysing agent has a strong and fast haemolysing activity and, additionally, high selectivity for red blood cells.
  • the inventive haemolysing agent is preferably soluble in volatile solvents, such as lower alcohols and acetonitril.
  • a device comprising a cavity for receiving a blood sample, said cavity containing a haemolysing agent in a dried form; the device being characterised in that said haemolysing agent comprises saponins originating from Quillaja saponaria Molina.
  • a method of producing a device of the above aspect of the present invention comprising introducing a solution of saponins from a purified Quillaja saponaria Molina extract into the cavity of the device; and allowing the solvent to evaporate, leaving the purified extract in a dried form in the cavity.
  • the Quillaja haemolysing agent used in devices for analysing components in blood samples according to the invention is obtained from the bark of the Quillaja saponaria Molina tree.
  • Raw extracts of this bark are commercially available as white powders including 7-15 % saponins (Merck and Acros).
  • the previously known purified Quillaja saponaria Molina extracts which are used as adjuvants in vaccines, should have low haemolysing activity, if any, in order not to be toxic.
  • the extract Before use in the devices according to the invention, the extract is subjected to a purification procedure, such as sequential extractions and/or sample displacement chromatography.
  • a purification procedure such as sequential extractions and/or sample displacement chromatography.
  • the purified saponin of the present invention is preferably dissolvable in a volatile solvent. This is a major advantage compared with currently used water soluble saponins, since the saponin of the invention dissolved in a volatile solvent easily and quickly may be dried through evaporation even in narrow spaces where the interface between the solution and the surrounding air is highly limited, such as in a capillary chamber, significantly reducing the speed of evaporation.
  • the saponin haemolysing agent of the invention is preferably present in the device in an amount sufficient to haemolyse any blood sample, received in a cavity or chamber of the device, within not more than 2 minutes of contacting the blood sample. For reason of not unduly influencing the properties of such a blood sample, such as by diluting it or introducing high amounts of substances foreign to the blood which may disturb analysis of the same, it is desirable to keep the amount of the haemolysing agent as low as possible, while still being able to haemolyse the blood sample sufficiently fast.
  • the device according to the invention may be designed depending on the analysis to be performed. In its simplest embodiment the device comprises a cavity for receiving a blood sample, which cavity contains a purified Quillaja saponin in dried form.
  • the cavity of the device is defined by two surfaces, which surfaces are preferably plane-parallel to each other.
  • the surfaces may be formed by two sheets of, e.g. plastic, which are joined together, directly or via spacers. If the sheets are joined directly, there may be depressions in one or both of them in order to enable the forming of the cavity.
  • the surfaces may be formed by moulding, such as injection moulding or other suitable technique.
  • the cavity of the device may have a predetermined fixed thickness, and may .completely or partly, define a predetermined volume, which provides a possibility to determine the count of biological components per volumetric unit, i.e. the component concentration, of the blood sample, through e.g. photometric analysis using a photometer or visual analysis using a microscope and/or a digital camera, wherein the photometer, or other equipment, is used to obtain data over a volume of the cavity defined by the cavity thickness and a predetermined area perpendicular to said thickness. An analysis is further facilitated if the surfaces of the cavity are plane-parallel, which is preferred, since the analysed volume is then more easily calculated.
  • the cavity is capillary, whereby a blood sample received and contained therein may be held without spilling despite the gravitational pull on the sample.
  • the device may further comprise an inlet, communicating the cavity of the device with any medium surrounding the device outside of said device.
  • the inlet is preferably capillary, thus enabling a blood sample to be sucked, by means of capillary action, into the cavity of the device. This is further facilitated by the cavity also being capillary, which is preferred. It may thus be possible for the device to directly acquire a blood sample, which is received by its cavity, simply by contacting the capillary inlet of the device with blood, since the blood will then be sucked into the capillary cavity by capillary action.
  • a convenient application, made possible with the device exhibiting these preferred features, is to use the device for obtaining a blood sample directly from, e.g. a pricked finger of a patient.
  • the invention is not limited to a single cavity.
  • a plurality of cavities, capillary or non-capillary may be preferred.
  • the device of the invention may be adapted to receive any type of blood sample in its cavity, such as whole blood, diluted blood, serum or plasma, or any other sample which may contain red blood cells (erythrocytes), from any donor, human or animal.
  • any type of blood sample in its cavity such as whole blood, diluted blood, serum or plasma, or any other sample which may contain red blood cells (erythrocytes), from any donor, human or animal.
  • red blood cells erythrocytes
  • the purified quillaja saponin is preferably first dissolved in an organic solvent, which is volatile at relatively low temperature.
  • suitable solvents are acetonitril and lower alcohols, preferably methanol, ethanol or isopropanol or mixtures thereof, methanol being the most preferred.
  • the obtained solution is then introduced into the device, which is preferably subjected to heating at a temperature sufficient for evaporating the solvent.
  • the solvent is methanol, which is preferred, the temperature should preferably be less than about 65 0 C, and preferably higher than about 45 0 C, although evaporation at room temperature may also be utilized. No expensive or complicated equipment is necessary, such as is the case with e.g.
  • the device including a haemolysing agent enables a haemolysing reaction within the device which makes the sample ready for analysis.
  • the haemolysis is initiated when the blood sample comes into contact with the haemolysing agent.
  • the device Since the haemolysing agent is provided in a dried form, the device may be transported and stored for a long time without affecting the usability of the device. Thus, the device with the haemolysing agent may be manufactured and prepared long before the introduction of a blood sample. As no manual addition or dosing of haemolysing agent is needed the device of the present invention may easily and reproducibly be used by even an untrained person, and not necessarily in a regular standardised laboratory environment. The device may thus form a ready-to-use kit in order to provide a haemolysed blood sample in a form ready to be analysed by any of the many analysis methods requiring haemolysed blood.
  • the raw saponin extract obtained (Merck or Acros) is further purified by the sample displacement chromatography method which was performed as follows:
  • the raw saponin extract is dissolved in water and the obtained water solution was diluted with 30%, by volume, methanol. This solution is applied on a "Reversed Phase C18" column. The column is washed with 30/70 methanol/water and the saponins are then displaced with 80/20 methanol/water until a stable base line is obtained. The combined saponins in methanol/water are air dried on stainless steel trays with HEPA filtered air.
  • the saponin powder is dissolved in methanol together with other reagents and additives adapted to the intended analysis to be performed.
  • This solution is subsequently introduced into the device, which is subjected to drying at a temperature of less than about 65 0 C for a period of less than 10 minutes, preferably 6-8 minutes, until the methanol has evaporated and the saponins and other reagents and additives are dried.
  • the obtained device is then ready for use.

Abstract

La présente invention concerne un dispositif utile pour la détermination de composants dans un échantillon sanguin. Le dispositif comprend une cavité destinée à recevoir ledit échantillon, cavité dans laquelle un agent d'hémolyse sous forme séchée est inclus. Ledit agent comprend des saponines provenant de Quillaja saponaria Molina.
PCT/SE2007/000747 2006-09-08 2007-08-24 Agent d'hémolyse WO2008030154A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
SE0601852-7 2006-09-08
SE0601852A SE0601852L (sv) 2006-09-08 2006-09-08 Hemolyseringsmedel
US90650407P 2007-03-13 2007-03-13
US60/906,504 2007-03-13

Publications (1)

Publication Number Publication Date
WO2008030154A1 true WO2008030154A1 (fr) 2008-03-13

Family

ID=39157491

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/SE2007/000747 WO2008030154A1 (fr) 2006-09-08 2007-08-24 Agent d'hémolyse

Country Status (1)

Country Link
WO (1) WO2008030154A1 (fr)

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3198064A (en) * 1961-06-29 1965-08-03 Welch Allyn Inc Blood sample holder
US3883425A (en) * 1973-12-10 1975-05-13 Wadley Res Inst & Blood Bank Detoxification of saponins
US4751179A (en) * 1984-05-31 1988-06-14 Coulter Electronics, Inc. Method and reagents for differential determination of four populations of leukocytes in blood
WO1988009336A1 (fr) * 1987-05-29 1988-12-01 Cambridge Bioscience Corporation Adjuvant a base de saponine
US5057540A (en) * 1987-05-29 1991-10-15 Cambridge Biotech Corporation Saponin adjuvant
US5278047A (en) * 1989-04-25 1994-01-11 Lilja Jan E Method of analysis, reagent composition and use thereof for glucose determination
WO1995009179A1 (fr) * 1993-09-30 1995-04-06 Seed Capital Investments (Sci) B.V. Composes presentant une activite d'adjuvant
WO2004092329A2 (fr) * 2003-04-08 2004-10-28 Galenica Pharmaceuticals, Inc. Analogues de saponine semi-synthetiques de transport et de stimulation immunitaire pour vaccins a adn et arn

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3198064A (en) * 1961-06-29 1965-08-03 Welch Allyn Inc Blood sample holder
US3883425A (en) * 1973-12-10 1975-05-13 Wadley Res Inst & Blood Bank Detoxification of saponins
US4751179A (en) * 1984-05-31 1988-06-14 Coulter Electronics, Inc. Method and reagents for differential determination of four populations of leukocytes in blood
WO1988009336A1 (fr) * 1987-05-29 1988-12-01 Cambridge Bioscience Corporation Adjuvant a base de saponine
US5057540A (en) * 1987-05-29 1991-10-15 Cambridge Biotech Corporation Saponin adjuvant
US5278047A (en) * 1989-04-25 1994-01-11 Lilja Jan E Method of analysis, reagent composition and use thereof for glucose determination
WO1995009179A1 (fr) * 1993-09-30 1995-04-06 Seed Capital Investments (Sci) B.V. Composes presentant une activite d'adjuvant
WO2004092329A2 (fr) * 2003-04-08 2004-10-28 Galenica Pharmaceuticals, Inc. Analogues de saponine semi-synthetiques de transport et de stimulation immunitaire pour vaccins a adn et arn

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