The present invention relates to a stain, a slide and a method of preparation of a blood sample.

Various types of cells and particles may be found in the blood of a human or animal, for example, red blood cells (RBCs) and several forms of white blood cells (WBCs) . In a healthy individual, the numbers of each type of cell may be controlled by complex body mechanisms. Any variation from the normal range can indicate serious physiological or pathological changes within the body. Therefore, it is important to be able to determine the number of each type of cell or particle in the blood. This may be achieved by preparation of a suitable sample of the blood or other biological fluid or material of a patient or plant and then screening the sample, for example under a microscope. The stain may be used to stain cellular components of plants, and in this specification "plants" should be taken as inclusive of fungi, bacteria and the like present in water and other environmental samples.

Conventionally, a sample may be prepared by first

preparing a blood smear on a slide, allowing the smear to dry in air, then dipping the smear in a bath of methanol or the like (to fix the cells) and finally staining the smear.

Conventional stains and staining methods may involve a series of complex and time consuming steps. As a result the whole staining process may take a considerable time to complete, for example may take an hour, and the operator should be skilled and experienced in order to obtain a satisfactory result. During the smearing process, cells and particles in the blood may not be distributed evenly or may be damaged and therefore become difficult or impossible to identify during screening.

In addition, many conventional stains, when the components are mixed, may only be stable for a short time, for example 2-3 days, and if purchased in bulk may easily become contaminated if not used quickly. Further, some stains may differentiate only certain blood cell types, and/or may only be effective in staining the blood cells of certain animal species.

In view of the problems and complexities outlined above, it is not convenient for samples to be prepared by these methods on a small scale or by a person who is not specialised in such techniques, for example in a surgery or a practice. As a result, blood is often sent to remote contract laboratories for analysis. This may be costly, require relatively large samples of blood to be supplied, and involve a considerable time delay. In addition, during transportation the blood may degenerate and the particles and cells therein may become damaged.

An object of the invention is to provide a means whereby a non-skilled person may produce good quality test specimens without difficulty.

In one aspect, the present invention provides a stain for animal or plant samples comprising the following components :-

(i) An acidic or basic buffer whose tonicity can be varied depending on the nature of the material to be examined;

(ϋ) A fixative and organic membrane modifier;

(iϋ) A compound which stains nuclear and cytoplasmic components in the sample; and

(iv) A solvent.

Preferably the buffer is Citric acid, Trisodium salt, dihydrate F. . 294.1 (Formula C ₆ H ₅ O _y Na ₃ .2H ₂ 0) .

Preferably, the membrane modifier is 2-Ethoxyethanol (Ethyleneglycolmonoethyl ether) F.W. 90.12 (Formula C ₂ H ₅ OCH ₂ CH ₂ OH) .

Preferably, the staining compound is Azure B (c.1.52010; Azure 1 Certified by BSC differentiating cellular RNA and DNA in plant tissue) F.W. 305.8. ( ^v Formula or a derivative of Azure B.

Preferably, the Azure B or derivative thereof has a dye content of approximately 80%.

Preferably, the solvent is deionised or distilled water.

Typically, the stain may be used to stain a sample of blood taken from a human or an animal, and may differentiate the types of cells and/or particles therein.

The stain of the present invention has the advantages of having good stability at room temperature, is able to give a good contrast between types of blood cells and intracellular structures, and may be effectively used to stain the blood of a variety of different animal species.

In another aspect, the present invention provides a method of staining comprising mixing a volume of liquid with a stain having the following components:

(i) An acidic or basic buffer whose tonicity can be varied depending on the nature of the material to be examined;

(ϋ) A fixative and organic membrane modifier;

(iϋ) A compound which stains nuclear and cytoplasmic components in the sample; and

(iv) A solvent;

and allowing the mixture to stand.

Preferably, the stain has the following components:

(i) Citric acid, trisodium salt, dihydrate F.W. 294.1 (Formula C ₆ H ₅ 0 _? Na ₃ .2H ₂ 0) ;

(ii) 2-Ethoxyethanol ( Ethyleneglycolmonoethyl ether) F.W. 90.12 (Formula C ₂ H ₅ OCH ₂ CH ₂ OH) ;

(iϋ) Azure B (c.1.52010; Azure 1 Certified by BSC differentiating cellular RNA and DNA in plant tissue) . F.W. 305.8 (Formula C ₁₅ H ₁₆ CIN ₃ S) ; and

(iv) Deionised and/or distilled water;

Preferably, the Azure B has a dye content of approximately 80%.

Typically, the liquid may be blood.

The method of staining of the present invention has the advantage that it consists of a quick and simple one stage process, and can be used to stain a fresh liquid blood sample.

Typically, the blood and the stain may be mixed in the ratio 1:4 and may be mixed by gentle agitation.

Preferably, the mixture is allowed to stand for between one minute and sixty minutes at a temperature of between 15°C and 60°C.

In yet another aspect, the present invention provides a slide adapted to draw liquid into itself by capillary action.

Typically, the liquid may be a sample of stained or unstained blood.

An advantage of the slide of the present invention is that a good quality monolayer blood film may be produced without the need for smearing, since a drop of blood may simply be introduced to one end of the

capillary chamber and allowed to flow by capillary action along the length of the chamber to form a monolayer of cells. Therefore the slide of the present invention is easy to use and facilitates the quick and simple preparation of a fresh blood sample which may then be analyzed in the conventional way.

Preferably, the slide comprises a base and a cover placed together to form a capillary chamber.

A further advantage of a slide in accordance with the invention is that the capillary chamber provides for a controlled or known volume of test sample, such as blood, to be examined, thereby enabling quantative analysis. Conventional slides are useful for qualative analysis only.

Preferably the base and the cover are made of a glass and/or a plastic material. Preferably, the glass is a very flat glass, such as float glass.

Typically, the cover may be fixed to the base using an adhesive which may act as a sealant of the capillary chamber.

During manufacture, microspheres may be used to separate the cover from the base, thereby ensuring the gap between the cover and the base is of a known distance. The microspheres may be between 5 and 20 microns in diameter and made of Vinyl-Benzene and/or polystyrene. The microspheres are preferably contained in an aqueous solution, and provided that they are not soluble in the adhesive, may be mixed with the adhesive prior to application on the base or cover.

The capillary chamber may be defined by providing

grooves in the base, which act to prevent the flow of the adhesive into the chamber, which preferably is of controlled volume.

Optionally the glass or plastics base and or cover may be treated or coated to assist or retard the flow of the test sample within the slide.

Optionally the slide comprises a base made of plastics, either by an extrusion or moulding process, the base having a concave or recessed portion of known volume, and a flat or planar cover.

In a fourth aspect, the present invention provides a method of preparation of a blood sample that comprises the steps of staining a quantity of blood and introducing the stained blood to a slide which draws the stained blood into itself by capillary action.

The method of preparation of the present invention has the advantage of being quick and easy, producing a good quality monolayer blood film, for example to facilitate blood cell counts. No special skills or techniques are necessary, and the method is convenient for use on a small scale, for example in a general practitioner's surgery. Further, only a small amount of blood is necessary, therefore reducing trauma to small animals when blood is taken.

In a final aspect, the present invention provides a kit comprising a stain having the following components:

(i) An acidic or basic buffer whose tonicity can be varied depending on the nature of the material to be examined;

(ii) fixative and organic membrane modifier;

(iii) A compound which stains nuclear and cytoplasmic components in the sample; and

(iv) A solvent,

and a slide adapted to draw liquid into itself by capillary action.

EXAMPLE

A stain according to the present invention is prepared in the following way:

Trisodium citrate (16.0g) is dissolved in distilled water and made up to 1.01 (equivalent to 50% isotonicity for mammalian red blood cells). 2-Ethoxyethanol (200ml) and Trisodium citrate solution (800ml) are mixed by stirring. (This is the primary buffer solution.) pH of buffer (unadjusted) is 8.9. Azure B (l.Og) is added to 500ml of primary buffer and made up to 1.01. (This is the working stain). The working stain is filtered under gravity through Watmans 113V wet strengthened filter paper.

A sample of blood is taken from an animal and is added to the stain in the ratio of 1 part blood to 4 parts stain. The blood and the stain are mixed by gentle agitation, and the mixture is allowed to incubate for between 1 minute and 60 minutes at a temperature of between 15°C and 60°C.

Embodiments of the present invention will now be described by way of example with reference to the accompanying drawings, in which:

Figure 1 depicts a side elevation of a preferred embodiment of a slide in accordance with the present invention;

Figure 2 is a top view the slide shown in Figure 1;

Figure 3 is an side view of an alternative embodiment of slide of Figure 3; and

Figure 4 is a top view of the slide of Figure 3.

Referring firstly to Figures 1 and 2, the slide 1 has a base 2 and a cover 3, both being manufactured from a glass material. The dimensions of the base 2 and the cover 3 are 25mm x 40mm x 1.1mm and 15mm x 5mm x 0.3- 0.6mm, respectively.

The slide 1 is also provided with microspheres 4, which act as spacers situated along the side edges of the cover 3. These spacers have a thickness of approximately 5.0μm, but in other embodiments of the invention the spacers may have a thickness of, say, between 4.0 and 25.0μm.

The cover 3 is attached to the base 2 by an adhesive 5 along the edges of the cover 3 and the sides of the spacers thus forming a capillary chamber 6. Grooves 7 are provided to prevent the flow of adhesive 5 into the capillary chamber 6.

A drop (1.0 - 5.0μl) of the stained blood (not shown), is introduced to either of the openings 8 of the capillary chamber 6.

The blood is then drawn into the capillary chamber 6 by capillary action, forming a fine layer between the

cover 3, the base 2 and the spacers. The adhesive strips 5 act as a sealant of the capillary chamber 6 to prevent leakage.

The slide 1 containing the stained blood is then analysed by any suitable method.

In Figures 3 and 4, the base 2 is made of a plastics moulding. The capillary chamber 6 is thus provided by a recess formed as part of the moulded base 2. The cover 3 may be glass or plastics. An adhesive or tape 5 is shown to fix the cover 3 to the base 2.