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
  • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
• • 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

Definitions

• the present invention relates to a stain, a slide and a method of preparation of a blood sample.
• red blood cells RBCs
• WBCs white blood cells
• 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.
• 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.
• 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.
• An object of the invention is to provide a means whereby a non-skilled person may produce good quality test specimens without difficulty.
• the present invention provides a stain for animal or plant samples comprising the following components :-
• the buffer is Citric acid, Trisodium salt, dihydrate F. . 294.1 (Formula C 6 H 5 O y Na 3 .2H 2 0) .
• the membrane modifier is 2-Ethoxyethanol (Ethyleneglycolmonoethyl ether) F.W. 90.12 (Formula C 2 H 5 OCH 2 CH 2 OH) .
• 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.
• the Azure B or derivative thereof has a dye content of approximately 80%.
• the solvent is deionised or distilled water.
• 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.
• the present invention provides a method of staining comprising mixing a volume of liquid with a stain having the following components:
• the stain has the following components:
• Citric acid trisodium salt, dihydrate F.W. 294.1 (Formula C 6 H 5 0 ? Na 3 .2H 2 0) ;
• the Azure B has a dye content of approximately 80%.
• 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.
• the blood and the stain may be mixed in the ratio 1:4 and may be mixed by gentle agitation.
• the mixture is allowed to stand for between one minute and sixty minutes at a temperature of between 15°C and 60°C.
• the present invention provides a slide adapted to draw liquid into itself by capillary action.
• 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.
• 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.
• the base and the cover are made of a glass and/or a plastic material.
• the glass is a very flat glass, such as float glass.
• the cover may be fixed to the base using an adhesive which may act as a sealant of the capillary chamber.
• 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.
• 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.
• 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.
• 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.
• the present invention provides a kit comprising a stain having the following components:
• 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.
• 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.
• Figure 4 is a top view of the slide of Figure 3.
• 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 5.0 ⁇ m, but in other embodiments of the invention the spacers may have a thickness of, say, between 4.0 and 25.0 ⁇ m.
• 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 - 5.0 ⁇ l) 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.
• 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.

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• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Engineering & Computer Science (AREA)
• Biomedical Technology (AREA)
• Molecular Biology (AREA)
• Immunology (AREA)
• Chemical & Material Sciences (AREA)
• General Physics & Mathematics (AREA)
• Urology & Nephrology (AREA)
• General Health & Medical Sciences (AREA)
• Analytical Chemistry (AREA)
• Physics & Mathematics (AREA)
• Pathology (AREA)
• Biochemistry (AREA)
• Hematology (AREA)
• Cell Biology (AREA)
• Microbiology (AREA)
• Biotechnology (AREA)
• Food Science & Technology (AREA)
• Medicinal Chemistry (AREA)
• Investigating Or Analysing Biological Materials (AREA)
• Sampling And Sample Adjustment (AREA)

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Publications (1)

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Cited By (5)

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• 1995
  • 1995-09-06 GB GBGB9518129.3A patent/GB9518129D0/en active Pending
• 1996
  • 1996-09-05 AU AU68841/96A patent/AU6884196A/en not_active Abandoned
  • 1996-09-05 CA CA 2230889 patent/CA2230889A1/en not_active Abandoned
  • 1996-09-05 JP JP9510980A patent/JPH11515095A/ja active Pending
  • 1996-09-05 EP EP96929428A patent/EP0848818A1/de not_active Withdrawn
  • 1996-09-05 WO PCT/GB1996/002192 patent/WO1997009616A1/en not_active Application Discontinuation

Non-Patent Citations (5)

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