WO2008030154A1

WO2008030154A1 - Haemolysing agent

Info

Publication number: WO2008030154A1
Application number: PCT/SE2007/000747
Authority: WO
Inventors: Bertil Johnny Ingemar Svensson; Lena Margaretha Svensson
Original assignee: Hemocue Ab
Priority date: 2006-09-08
Filing date: 2007-08-24
Publication date: 2008-03-13

Abstract

The present invention concerns a device useful for the determination of components in a blood sample. The device comprises a cavity for receivin the blood sample, in which cavity a haemolysing agent in a dried form is included. The haemolysing agent comprises saponins originating from Quillaja saponaria Molina.

Description

HAEMOLYSING AGENT

Field of the invention

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.

Background art

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. 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.

One group of compounds known to have haemolysing activity is saponins. 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.

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. In order to prepare a device for this glucose determination 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. In order to obtain a satisfactorily dried reagent in the device, so that reproducible quantitative determinations of glucose can be made, 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.

A problem to be solved by the present invention was to find a haemolysing agent, a solution of which can be dried, within an analysis device, in a less elaborate way. Additionally 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. In view of the urgent need to get fast analysis results in hospitals and medical centres there is also an interest in haemolysing agents which haemolyse not only efficiently but also rapidly.

Summary of the invention

According to the present invention it has been found that 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. Also, the inventive haemolysing agent is preferably soluble in volatile solvents, such as lower alcohols and acetonitril.

Thus, in one aspect of the present invention, there is provided 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.

In another aspect of the present invention, there is provided a method of producing a device of the above aspect of the present invention, the method 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.

In yet another aspect of the present invention, there is provided a for the use of a dried and purified Quillaja saponaria Molina extract as a haemolysing agent in devices for the determination of components in blood. Detailed description of the invention

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).

In order to obtain reproducible quantitative determinations of components in the blood sample to be analysed it may be necessary to subject these powders to purification to such an extent that a highly purified saponin haemolysing agent is obtained.

In contrast to the purified Quillaja saponaria Molina extracts which are used in the devices according to the present invention, 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.

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.

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.

According to one embodiment 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. Alternatively, the surfaces may be formed by moulding, such as injection moulding or other suitable technique.

Further, 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. Preferably the cavity is capillary, whereby a blood sample received and contained therein may be held without spilling despite the gravitational pull on the sample.

To facilitate the introduction of a blood 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.

It should be noted that the invention is not limited to a single cavity. For several applications, 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.

One type of analysis where preliminary tests have indicated that the Quillaja saponin give reproducible and highly satisfactory results is disclosed in the co-pending application PCT/SE2006/000311. This application, which by this reference is incorporated in its entirety in the present application, concerns the analysis of white blood cells. Devices suitable for the analyses are also disclosed in this publication. The devices according to the present invention are however not limited to this type of analysis, but other analyses requiring other types of reagents and/or other types of detection systems, e.g. photometric, are also included.

When preparing the device including the dried haemolysing agent the purified quillaja saponin is preferably first dissolved in an organic solvent, which is volatile at relatively low temperature. Examples of 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. When the solvent is methanol, which is preferred, the temperature should preferably be less than about 65⁰C, and preferably higher than about 45⁰C, although evaporation at room temperature may also be utilized. No expensive or complicated equipment is necessary, such as is the case with e.g. evaporation under vacuum or freeze drying. 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. Thus, there is no need for haemolysing the sample manually, which simplifies the process of obtaining a haemolysed blood sample in the device, making the device especially suitable for use directly in an examination room while a patient is waiting.

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 invention is further illustrated by the following non-limiting example.

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.

When a device according to the invention is prepared, 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 65⁰C 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.

Claims

1. A device comprising a cavity for receiving a blood sample, said cavity containing a haemolysing agent in a dried form; c h a r a c t e r i s e d in that said haemolysing agent comprises saponins originating from Quillaja saponaria Molina.

2. A device according to claim 1 wherein the haemolysing agent is purified by sequential extractions.

3. A device according to claim 1 wherein the haemolysing agent is purified by sample displacement chromatography.

4. A device according to any one of the claims 1-3 wherein the haemolysing saponins are soluble in a lower alcohol or acetonitril.

5. A device according to claim 4 wherein the alcohol is selected from the group consisting of methanol, ethanol and isopropanol.

6. A device according to any one of the preceding claims wherein the device includes additional reagents and additives.

7. A method of producing a device according to claim 1 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.

8. Use of a dried and purified Quillaja saponaria Molina extract as a haemolysing agent in devices for the determination of components in blood.