WO1999063324A1

WO1999063324A1 - A method and a device for preparing a biological specimen

Info

Publication number: WO1999063324A1
Application number: PCT/SE1999/000830
Authority: WO
Inventors: Christer FÅHRAEUS; Liana SANDSTRÖM
Original assignee: Cellavision Ab
Priority date: 1998-05-15
Filing date: 1999-05-14
Publication date: 1999-12-09
Also published as: AU4540299A; EP1078240A1

Abstract

In a method for preparing a biological specimen, e.g. by means of staining, fluids which are to be applied to the specimen are atomised with the aid of ultrasound prior to their application. In this way, small drops are obtained which are gentle to the specimen and which are able to prepare the whole specimen quickly and with only a small amount of fluid being required. Between the application of the different fluids the specimen is spun for removing excess fluid so that no mixing occurs of successively applied fluids. A device for carrying out the method is also disclosed.

Description

A METHOD AND A DEVICE FOR PREPARING A BIOLOGICAL SPECIMEN

Field of the Invention

The present invention relates to a method for preparing a biological specimen, comprising the step of applying at least one fluid to the specimen. The inven- tion also relates to a device for carrying out the method. Background of the Invention

In the health and medical care services, it is common to take specimens of body fluids or tissue and subse- quently study the specimens in a microscope or by means of some other optical method. In order to make it possible to study the specimens in a microscope they must usually be arranged in a thin layer or a monocellular layer (sometimes called a monolayer) on a slide and sub- sequently be stained to make the constituents, such as cells and cell structures, which one wishes to study clearly visible.

An example of the above is the so-called differential white cell count, in which the relative distribu- tion of white blood cells among a number of main types is determined.

Traditionally, the differential white cell count is carried out entirely manually. A blood specimen is taken from a patient whose blood is to be analysed. A few drops of the specimen are placed on a slide and are spread in a thin layer over the surface of the slide with the aid of an oblique, smaller slide. Subsequently, the specimen is fixed on the slide with the aid of a fixative.

In the next step, staining of the blood on the slide is carried out. For this purpose, the standard method used in Sweden is the so-called May-Grunwald-Giemsa method, in which the specimen is stained with the aid of the stains eosin and tiazin (one or more members of the group azureA, azureB, azureC, and methylene blue) . In this connection, the specimen is immersed in a solution of these stains, which is allowed to take effect for 20-45 minutes. After rinsing, the preparation of the specimen is complete. When the specimen is to be studied, the slide is placed in a microscope. Subsequently, a laboratory technician counts the white blood cells within a suitable area on the slide and determines the main type to which each of these cells belongs.

Other specimens, such as cervical smears, are prepared in a similar manner on a slide, i.e. they are fixed, stained, and rinsed by hand to prepare them for microscopy. This manual preparation of specimens is time-consuming and requires a large number of staff. Moreover, it is difficult to achieve high reproducibility . Consequently, there have been attempts to automate certain steps and to reduce the time spent on other steps. For example, it is known to carry out the spreading step in a spinner, in which case the blood specimen is placed on the edge of a slide which is spun at high speed so that the blood is spread over the surface of the slide. Automatic image processing of the specimens has also been tried.

Furthermore, staining kits have been developed in which the staining with eosin and tiazin is carried out sequentially. This results in faster absorption of the stains by the cells and, consequently, the stains do not need as long to take effect as in the May-Grύnwald-Giemsa method, in which they are mixed in a solution. However, the specimens are essentially handled using the same immersion technique as was previously used.

Another drawback of the prior art preparation methods is that they require fairly large amounts of staining solution. In order to economise on solutions, the same solution is generally used for many specimens, perhaps as many as a hundred. This results in poorer reproducibility. Moreover, there is a risk that cells from a specimen may come loose and get into the solution. As a result, the specimen from which the cells have come loose becomes less reliable and, moreover, the cells which have come loose can become attached to a specimen which is subsequently immersed into the solution. In that case, the result of the analysis of the latter specimen may also be incorrect. A further drawback of the known preparation methods is that the laboratory staff need to handle relatively large amounts of solutions, which in some cases may be unsanitary. In addition, the solutions must be dealt with in a suitable manner after staining so that they will not harm the environment.

Summary of the Invention

One object of the present invention is to provide a method for preparing a biological specimen by which the above problems are eliminated or at least diminished. Another object of the invention is to provide such a method which produces results of sufficiently high quality to permit both automatic image processing and manual microscopy of the specimen.

A further object of the invention is to provide a device for carrying out the above method.

These objects are achieved by a method according to claim 1 and a device according to claim 16. Preferred embodiments of the invention are stated in the subclaims . More specifically, a method for preparing a biologi- cal specimen comprises the step of applying at least one fluid to the specimen, the method being characterised by the step of atomising said fluid prior to applying it to the specimen.

An advantage of atomising the fluid prior to apply- ing it to the specimen is that it allows the shortening of the time required for the fluid to take effect after it has been applied. This is believed to be due to the fact that it is easier for the cells to absorb the fluid in atomised form through the cell membranes.

Another advantage of the atomisation is that much smaller amounts of the different fluids are required than when the specimen is immersed into the fluids according to the prior art. By being atomised, the fluid will spread like a mist over the specimen, which is advantageously arranged in a horizontal position, resulting in very good coverage of the specimen. Moreover, fresh fluid is always used on the specimens, which thus improves their quality.

A further advantage is that the atomised solution appears to be gentler to the specimen and, consequently, provides better quality stained specimens. For example, fewer cell parts are washed away with the atomised solution, which may be due to the fact that there is less excess fluid on the specimen surface than when the specimen is immersed into the fluid.

Yet another advantage is that the atomised fluid provides good wetting of the surface, since it does not collect in large drop formations.

In this connection, it should be noted that, in this case, atomising refers to fluid being broken into drops having an average diameter of about 10-100 μm, preferably 20-60 μm. Preferably the diameter of as many as possible of the drops, and in any case 50% of the drops, is within said range .

Furthermore, preparing a specimen refers to any preparation which can make the specimen suitable for analy- sis. Arranging the specimen on a slide may form part of the preparation, but it is not a requirement.

In an advantageous embodiment of the invention, the fluid is atomised essentially without the fluid accelerating towards the specimen. A certain small degree of acceleration is necessary for the drops to come loose from the nozzle, but it is advantageous to keep the acceleration at a low level so as to avoid the risk of the drops breaking structures, such as cells, of the specimen when striking against the specimen.

For the same reason, it is advantageous to let the atomised fluid fall freely over the specimen and not accelerate it towards the specimen after atomisation. In an advantageous embodiment of the invention, fluid is atomised with the aid of ultrasound. The ultrasonic technique has the advantage that it can atomise the fluid without accelerating it towards the specimen. Other known techniques for atomising fluids, such as atomisers, must utilise air pressure to achieve the atomisation. This means that there is a risk that drops may be accelerated in the direction of the specimen and that these drops break structures, such as cells, on the specimen when they strike against the specimen. A further advantage of the ultrasonic technique is that it is possible to control the size of the drops by the choice of frequency of the ultrasound and that it thus is possible to obtain drops within a desirable range. Subsequent to the application of fluid to the specimen, the specimen is suitably spun to remove excess fluid from the same. The fact that no fluid which can continue to act upon the specimen remains after spinning results in higher reproducibility in connection with the prepa- ration of specimens. In this context, spinning refers to placing a slide upon which the specimen is located in a horizontal position and rotating it about an axis extending perpendicular to the surface upon which the specimen is located so that the fluid on the slide is moved towards the periphery of the slide. It should be noted that the expression centrifuging is sometimes used instead of spinning.

The fluid which is applied to the specimen can be any fluid which is to be applied to the specimen in order to prepare the latter to make it suitable for analysis in a microscope or in some other instrument . A common method of preparing a specimen is to stain some structures in the specimen so that they can be studied. In connection with the staining, a fixative is usually first applied in order to fix the specimen, fol- lowed by at least one staining fluid, and finally a rinsing fluid to finish the staining. In an advantageous embodiment of the invention each of these fluids is atomised prior to being applied to the specimen. The atomisation and application are suitably carried out in the man- ner described above, i.e. without acceleration of the fluid towards the specimen in connection with the atomisation and by the atomised fluid being allowed to fall freely over the specimen. Suitably, the specimen is spun subsequent to the application of each fluid so that the excess of the most recently applied fluid is removed. In this way, dilution of the subsequent fluid with the excess of the previous fluid is also avoided, which is particularly important when the fluids are applied to the specimen in the small amounts that are involved when the fluids are atomised. Furthermore, by spinning away excess fluid, reaction between two successively applied fluids is minimised. For example, in connection with the preparation of blood specimens, it has been observed that when a tiazin solution is mixed with an eosin solution, tiazineosin precipitates occur on the blood cells, which is perceived as an inconvenience in connection with microscopy. If the fixative contains tiazin for prestain- ing the specimen, such precipitates can also occur if the fixative is mixed with eosin solution. The problem is particularly noticeable when the specimens are immersed in the different solutions, and it is known from the May-Grύnwald-Giemsa method. The spinning away also results in the subsequent step being made more efficient since there is no competition between the molecules in the different fluids but they are allowed to take effect on their own, thus increasing the speed of staining. Finally, it has been found that the rinsing is more effi- cient if the excess of the previous staining solution is spun away prior to that step.

It may also be suitable to spin the specimen during the actual application of an atomised fluid since this results in a more even distribution of the fluid over the specimen.

The specimen should be spun at a speed such that cells in the specimen which is being prepared are not subjected to an acceleration greater than 200 g. In the case of a specimen surface having a diameter of 20 mm this corresponds to a maximum speed of 4500 rpm.

An advantageous embodiment of the invention further comprises the step of placing the specimen on a circular slide arranged in a holder, in which an absorbent for absorbing excess fluid extends around the slide. Such a slide is described in SE 9601404-8. In connection with the spinning of the specimen, excess specimen and excess fluid are moved towards the periphery of the slide and are absorbed by the absorbent. In this way, the specimen and the fluid can be applied without soiling the surrounding area. Furthermore, the specimen can be arranged in the centre of the slide, making it easier to obtain a monolayer .

It is assumed that the above method functions well for any type of biological specimen. Preferably, it is usable for histological , cytological, and microbiological specimens. The method has been tested specifically and been found to function well with respect to blood specimens . Moreover, the method is particularly suitable for specimens which are to be analysed with the aid of automatic image processing since the method permits quick and easy parameter changes so that the sharp adjustment of contrasts required in automatic image processing can be easily carried out. Examples of cell structures which can be identified in connection with such automatic image processing of blood specimens include: nuclei, granules, chromatin structures, cytoplasm, white blood cells, auer rods, malaria plasmodia, intracellular parasites, nucleoli, vacuoles, bodies, Jolly bodies, so-called drumsticks, and other intracellular organelles. The invention also relates to a device for preparing a biological specimen, comprising preparation means for applying at least one fluid to the specimen. The device is characterised in that the preparation means comprise an atomiser nozzle for atomising the fluid prior to applying it to the specimen.

The same advantages are achieved by this device as the ones described above with respect to the method.

The atomisation and application to the specimen are preferably carried out in the same manner as described above. In an advantageous embodiment, the device comprises a receptacle for said fluid and supply means for supplying said fluid to the atomiser nozzle, the receptacle and the supply means being arranged in such manner that the fluid is supplied to the atomiser nozzle by gra- vity only. The fluid thus is supplied to the atomiser nozzle without utilisation of air pressure, pumping or other means which may result in acceleration of the fluid towards the specimen in connection with the atomisation. Brief Description of the Drawings The present invention will now be described by way of an example with reference to the accompanying drawings, in which

Fig. 1 is a schematic view of an embodiment of a device according to the invention which is usable for preparing a blood specimen; and

Fig. 2 is a flowchart of an embodiment of a method according to the invention relating to the preparation of a blood specimen.

Description of a Preferred Embodiment The following example relates to the preparation of a blood specimen. As shown in Fig. 1, in this instance, a preparation device comprises a centrifuge (sometimes called a spinner) 1. In the centrifuge there is a space 2, in which a slide 3 is to be horizontally located, as indicated by a dashed line. The space is designed so as to act as a holder holding the slide during centrifuga- tion. When the centrifuge is activated, it rotates the slide at high speed about an axis A, whereby any excess fluid on the surface of the slide is moved towards the edges of the slide and is removed from the slide. Centrifuges of this type are commercially available. One example is a centrifuge with the brand name "Horizonten" from Hettich Zentrifugen, Tuttlingen, Germany.

In this embodiment, the device further comprises a preparation unit P containing four receptacles 4 for different preparation fluids, one being intended for fixa- tive, one for eosin solution, one for tiazin solution, and one for rinsing fluid. Each receptacle 4 is connected by the intermediary of supply means in the form of a tube 5 to an ultrasonic nozzle 6, which is used for atomising the respective fluids. In each tube there is a valve 7 for opening and closing the tube. The receptacles

4 are arranged above the nozzles so that the fluid can be transported to the nozzles by gravity only.

Finally, the device comprises a control unit 8 for controlling the centrifuge 1 and the preparation unit P, its connection to these units being highly schematically shown. The control unit controls the start-up and the shut-off of the centrifuge 1, the opening and closing of the valves 7 and the activation and the shut-off of the ultrasonic nozzles 6. The control unit 8, which may con- sist of a suitably programmed computer, also enables fully automatic preparation of specimens.

A description of how a blood specimen can be prepared according to a preferred embodiment of the invention is given below, with reference to the flowchart in Fig. 2.

40 μl of blood are pipetted in the centre of a circular slide 3 having a diameter of 39 mm which has been placed in the centrifuge 1. The slide with the blood is spun for 2 s at a speed of 4500 rpm, whereby a monolayer is obtained over a major part of the surface of the slide (step 201 in Fig. 2) . It is desirable for the monolayer area to have a diameter of at least 20 mm so that it will contain a sufficient number of cells which can be analysed.

Subsequently, a staining kit from Wescor Inc., 459 South Main Street, Logan, Utah 84321, USA is employ- ed. The kit contains a fixative, Basofix Pre-Dip SS-049P, a first staining solution, Eosin red SS-049C, a second staining solution, Methylene blue SS-035B, and a rinsing fluid, Basofix Rinse SS-049A. The working times recommended by the manufacturer, with respect to immersion of the specimen in the solutions, are 100 s for the fixative, 33 s for each of the eosin solution and the methylene blue solution, and 30 s for the rinsing fluid, i.e. 196 s in total. In this example, the solutions are instead atomised prior to being applied to the specimen and the specimen is spun subsequent to each application of a solution so that any excess solution is removed.

More specifically, the blood specimen is first fixed with the aid of the fixative Basofix Pre-Dip, SS-049P (step 202) . The solution is atomised with the aid of one of the ultrasonic nozzles 6, whereby a "mist" is obtained which falls down on the specimen. An ultrasonic nozzle which has been tested and which can be used for the atomisation is the ultrasonic nozzle US1 from Lechler GmbH in Germany. With this nozzle, an average diameter of the drops of 36 μm is achieved. The fixative is allowed to take effect for 10-25 s, preferably 15 s, after which excess fixative is spun away for 1 s at a speed of 1000 rpm (step 203) . It should be mentioned that the fixative contains the stain azureB which provides a cer- tain amount of pre-staining of the specimen.

In the next step, the specimen is stained with an Eosin red, SS-049C (step 204) . In the same way as in the previous step, the solution is atomised and applied to the blood specimen, where the solution is allowed to take effect for 10-60 s, preferably 30 s, after which the specimen is spun for 1 s at a speed of 1000 rpm to remove excess solution (step 205) .

In the next step, the specimen is stained with the methylene blue solution SS-035B (step 206) which is also atomised and applied to the blood specimen, where it is allowed to take effect for 20-60 s, preferably 30 s, after which excess solution is spun away for 1 s at a speed of 1000 rpm (step 207) .

In the final step, the rinsing fluid Basofix SS-049A is atomised (step 208) and is applied to the blood specimen, where it is allowed to take effect for 10-30 s, pre- ferably 25 s. Subsequently, the specimen is spun for 1 s at a speed of 3000 rpm. The purpose of the higher spinning speed subsequent to the application of the final fluid is to dry the specimen.

The specimen has now been prepared and is ready to be analysed in a microscope or with the aid of automatic image processing.

In the above example, the total preferred time for the preparation solutions to take effect is 104 s, i.e. a substantially shorter time than in the traditional immersion method. Furthermore, the total amount of solution used in this example is 500-800 μl, while in the traditional immersion method about 10 times more solution is required for the equivalent staining. In the assessment made by experienced laboratory technicians, the result of the staining is as good as in connection with traditional immersion staining.

In the example described above, blood is pipetted onto a slide and spun, after which the staining is immediately carried out. The staining can also be carried out on specimens which in an earlier stage have been arranged in a monolayer on a slide and which subsequent- ly have been allowed to air dry or have been prefixed in methanol .

Claims

1. A method for preparing a biological specimen, comprising the step of applying at least one fluid to the specimen, c h a r a c t e r i s e d by the step of atomising said fluid prior to applying it to the specimen.

2. A method according to claim 1, wherein the step of atomising the fluid is carried out essentially without accelerating the fluid towards the specimen.

3. A method according to claim 1 or 2 , wherein the step of applying said fluid to the specimen comprises letting the atomised fluid fall freely over the specimen.

4. A method according to any one of claims 1-3, wherein the step of atomising said fluid comprises breaking the fluid into drops with an average diameter of about 10-100 ╬╝m, preferably about 20-60 ╬╝m.

5. A method according to any one of the preceding claims, wherein said fluid is atomised with the aid of ultrasound.

6. A method according to any one of the preceding claims, further comprising the step of spinning the specimen subsequent to the application of fluid to remove excess fluid.

7. A method according to any one of the preceding claims, wherein said fluid is at least one of a fixative, a staining fluid and a rinsing fluid.

8. A method according claim 1, wherein the step of applying at least one fluid to the specimen comprises first applying a fixative, subsequently applying at least one staining fluid and then applying a rinsing fluid, each of the fixative, the staining fluid, and the rinsing fluid being atomised prior to being applied to the speci- men .

9. A method according to claim 8, wherein the specimen is spun subsequent to the application of each of the fixative, the staining fluid, and the rinsing fluid.

10. A method according to any one of the preceding claims, wherein the specimen is spun during the application of said at least one fluid.

11. A method according to any one of claim 6, 9 or 10, wherein the specimen is spun in such a way that the acceleration is less than 200 g.

12. A method according to any one of the preceding claims, wherein said biological specimen is a histolo- gical, or a cytological, or a microbiological specimen, preferably a blood specimen or a cervical smear.

13. A method according to claim 1, wherein the biological specimen is a blood specimen and wherein a fixative is atomised and applied to the specimen and is allowed to take effect for a predetermined length of time, wherein the specimen is subsequently spun, wherein an eosin solution is subsequently atomised and applied to the specimen and is allowed to take effect for a predetermined length of time, wherein the specimen is subsequently spun, wherein a tiazin solution is subsequently atomised and applied to the specimen and is allowed to take effect for a predetermined length of time, wherein the specimen is subsequently spun and wherein a rinsing fluid is subsequently atomised and applied to the specimen which is subsequently spun.

14. A method according to claim 13, wherein the fixative is allowed to take effect for about 10-25 s, the eosin solution for about 10-60 s, the tiazin solution for about 20-60 s, and the rinsing fluid for 10-30 s.

15. A method according to any one of the preceding claims, further comprising the step of placing the specimen on a circular slide arranged in a holder, in which an absorbent for absorbing excess fluid extends around the slide .

16. A device for preparing a biological specimen, comprising preparation means (4-7) for applying at least one fluid to the specimen, c h a r a c t e r i s e d in that the preparation means comprise at least one atomiser nozzle (7) which is adapted to atomise the fluid prior to applying it to the specimen.

17. A device according to claim 16, wherein the device is adapted to atomise the fluid essentially without accelerating it towards the specimen.

18. A device according to claim 16 or 17, wherein the atomiser nozzle is an ultrasonic atomiser nozzle.

19. A device according to any one of claims 16-18, further comprising a receptacle (4) for said fluid and supply means for supplying said fluid to the atomiser nozzle (7) , the receptacle and the supply means being arranged in such manner that the fluid is supplied to the atomiser nozzle by gravity only.

20. A device according to any one of claims 16-19, further comprising spinning means (1) adapted to spin the specimen.

21. A device according to any one of claims 16-20, further comprising a holder (2) for supporting a specimen carrier, on which said specimen is arranged horizontally under the preparation means.

22. A device according to any one of claims 16-21, wherein the device is arranged in such manner that the atomised fluid falls freely over the specimen.