WO2006031095A1

WO2006031095A1 - Capillary-loading slide and method of microscopic research with correction of the serge silberberg effect

Info

Publication number: WO2006031095A1
Application number: PCT/NL2004/000629
Authority: WO
Inventors: Diarmaid H. Douglas-Hamilton; Jan P. W. Vermeiden
Original assignee: Leja Holding B.V.
Priority date: 2004-09-13
Filing date: 2004-09-13
Publication date: 2006-03-23

Abstract

A capillary loading slide (1) for microscopic research of a suspension of particulates, is disclosed which comprises a spaced apart top plate and bottom plate, said plates being joined to each other by a connecting layer (4) disposed between said plates, wherein the layer (4) and the plates together form a boundary surrounding a chamber (6) for microscopic measurements, the chamber (6) having an inlet (8) for introducing a suspension to be measured and an outlet (10) for purging air when the suspension is introduced, and laminar flow disturbers (12,14,x) are provided within the chamber (6) at fixed positions outside a measuring zone. Alternatively the area before the analysis zone is deepened forming a recess preventing the formation of a laminar flow. Furthermore a method of microscopic research is disclosed using the same capillary loading slide (1).

Description

Capillary-loading slide and method of microscopic research with correction of the Segre Silberberg effect

The invention relates to a capillary loading slide for microscopic research of a suspension of particulates, comprising a spaced apart top plate and bottom plate, said plates being joined to each other by a connecting layer disposed between said plates, wherein the layer and the plates together form a boundary surrounding a chamber for microscopic measurements, the chamber having an inlet for introducing a suspension to be analyzed and an outlet for purging air when the suspension is introduced, as well as a measuring zone for performing microscopic research. Such a capillary loading slide, hereinafter also referred to as ^λslide' , is known from the art, e.g. from US 6,551,554 in the name of the Applicant. The connecting layer typically comprises spacers within a curable matrix. The spacers predominantly determine the height of the chamber, as explained in detail in the cited patent. Such a slide has the advantage of providing a top and bottom plate - also referred to as cover slip and objective plate respectively - being permanently attached to each other at a specific chamber height, also referred to as the capillary diameter. The inner void of the slide or chamber, is particularly suitable for counting cells in biological materials such as sperm and blood samples. When examining sperm, for example, the aim is to establish how many spermatozoa are present in the sample and also how motile they are. When microscopic measurements are carried out using such a slide, some detrimental effects occur that are inherent to the use of a slide, in comparison to the gold standard for these measurements, which is a hemocytometer. A hemocytometer however has no fixed cover slip, it has to be cleaned each time, so that the measurement is more laborious in comparison to the use of a capillary loading slide which has a fixed cover slip. ^'The deviation of the research results of the disposable slide with a fixed cover slip compared to the results of the hemocytometer stems from a wide range of factors, most of which have to be taken into account in relation to the effect that a laminar flow arises within the suspension to be measured after it has been introduced into the chamber of a slide. The flow that develops within capillary systems is also referred to as the Poiseuille flow, and has a direction parallel to the plates of a capillary loading slide. The Poiseuille flow is zero near the capillary wall and maximum in the middle. Because of these different flow velocities, a transverse velocity gradient arises, which results in the transport of particles from the flow edges into faster-flowing layers of the fluid. This phenomenon is called the Segre Silberberg effect. It causes (cell) concentration changes within the chambered flow. Hereinafter this effect will be denoted as the SS effect. The SS effect has shown to be detrimental to a measurement of particles in a suspension in a capillary. Thus, a measurement from a capillary loading slide has to be corrected by a specific factor to obtain the ^Λtrue' concentration, which by convention is the concentration measured in a hemocytometer. Practice revealed that the correction factor depends -amongst others - on the diameter of the capillary (the chamber height) , the viscosity of the sample, the size and shape of the suspended particles or cells, the flow velocity of the fluid, surface tension, angle of contact between fluid and surface of the capillary, the distance from the inlet of the chamber where the assessment is performed and the extent to which a Poiseuille flow can develop in the chamber. The invention aims at solving the above problems that are related to the necessity of correcting the measurements in a capillary loading slide, in order to comply with the standard of a hemocytometer. One object of the invention is thus to eliminate the use of a correction factor, while maintaining the inherent advantages of a capillary loading slide.

In a first aspect, the capillary loading slide according to the invention therefore comprises laminar flow disturbers which are provided within the chamber at fixed positions outside the measuring zone. Such a disturber, or a disturbing element, typically has the form of a platelet. As such, the disturbers have the effect of opposing, blocking, or preventing the Poiseuille flow in a capillary so as to form a hindrance or expansion. In practice, the disturbers prove to be able to hinder the Poiseuille flow to such an extent that no SS effect correction factor is longer necessary for microscopic measurements, i.e. the gold standard of a hemocytometer is equaled by a measurement in a slide according to the invention. In addition, the slide has the advantage in comparison to the hemocytometer of a more effective use of a common 1Ox and 2Ox microscopic lens, because the focal depth through the sample is typically much smaller compared to a hemocytometer, which has a typical chamber height of 100 micrometer. In practice, a sample measured in a hemocytometer requires suspended particles to settle before a measurement is taken, especially in the case of spermatozoa which need to be even immobilized before an accurate counting can be done.

Capillary filled chambers with a chamber height of about 20 micrometer can be used both in manual and in computer automated semen analysis (CASA) . The use of a CASA system has the advantage that a much higher number of cells can be assessed and that motility patterns of sperm can be measured in such chambers. Twenty micrometer chamber height is within the focal depth of a 10x objective lens. Analysis can be started immediately after loading, and the motility patterns of living spermatozoa can be assessed. The analysis chamber has a fixed cover slip and has a chamber height of 20 micrometer. For the assessment of cell numbers a 20 micrometer height is convenient, but different chamber heights are possible as well, e.g. between 10 and 200 micrometer. For clarity, it is remarked that the measuring zone is the zone wherein the microscopic research is performed, i.e. the zone that is focused upon by the microscope. This zone typically has a surface dimension of 3.5 x 1.5 mm and a height that is herein referred to as the chamber height. These dimensions are not critical and other dimensions are possible. However, the actual assessments of the number of suspended particle or cells has to be performed within 0-1.5 mm after the area where the formation of laminar flow is prevented or where the laminar flow is disturbed.

The top and bottom plate are typically made of a material which transmits UV and/or visible light, preferably glass, and the top plate should be selected to be as thin as possible (0.1 -0.4 mm) in order to facilitate the examination under a microscope.

The connecting layer typically comprises a matrix of curable material, such as a resin, preferably with adhesive properties, in which particulates, or so called micro-beads are dispersed. These micro-beads serve as so-called spacers, as their maximum diameter determines the distance between the two plates that are joined to each other by the connecting layer. Advantageously, the curable resin is non-toxic and does not effect the functions of the cells to be studied. By preference, the capillary loading slide according to the invention comprises laminar flow disturbers which are at least connected to the top or the bottom plate. A connection of the disturbers to the plates provides a reliable attachment and is convenient for several types of disturbers as will be presented herein below. Furthermore, when the disturbers are connected to one of the plates, the process of assembling the slide has the additional advantage that the plates can be provided with disturbers in a separate step before the assembling step wherein the two plates are joined by the intermediate connecting layer. According to a preferred embodiment of the capillary loading slide according to the invention, the laminar flow disturbers comprise pillar disturbers that are connected to both the top plate and the bottom plate. As such, the pillar disturbers provide a hindrance over the whole height of the chamber which proves effective in disturbing the flow of a suspension after introduction in the slide.

In a further preferred embodiment of the capillary loading slide according to the invention, the laminar flow disturbers comprise projection disturbers that are connected to either of the top plate and the bottom plate and which have a height which is less than the height of the chamber between top plate and bottom plate. For clarity's sake, it is mentioned that projection disturbers are connected to the top plate or bottom plate only, i.e. an individual projection disturber is not connected to both plates, contrary to a pillar disturber which is connected to both plates. A projection disturber can more appropriately be called a finlet, because the height of the projection disturber is such that it leaves room between its top and the plate of the slide to which it is not connected. It proves that the projection disturber has a different characteristic in hindering the flow of a suspension after introduction, compared to a pillar disturber. However, also the projection disturber proves effective in disturbing the flow in order to fulfill the objective of the invention. Typically, the projection disturbers have such a height that the space between the opposing glass plate on the one hand and the top of the projection disturber on the other hand, leaves ample space for the passing of the suspended particles of cells in the suspension. Normally, the projection disturbers have a height that is half of the chamber height, or less.

Especially the combination of different types of disturbers, i.e. both pillar and projection disturbers, provides an enhanced disturbing effect of the Poiseuille flow that inherently is present in a sample that is introduced in the chamber. The enhancement of the disturbance is probably due to the different flow characteristics around the two different types of disturbers. The shape of the pillar and projection disturbers is such that they optimally disturb the laminar flow, allowing the formation of a vortex downstream of the disturber. In order to accomplish that goal, the shape of a pillar in cross section can for instance be round, square, or polygonal. In a still further preferred embodiment of the capillary loading slide according to the invention, the laminar flow disturbers are made of a resin. Resins are convenient for forming these structures. In addition and advantageously, the resin used for a pillar disturber comprises spacers, such as micro-beads. As such, the same spacer, i.e. maximum diameter of micro-beads can be used as comprised in the connecting layer of the slide, so that the plates are uniformly distanced. Preferably, the laminar flow disturbers of the capillary loading slide according to the invention comprise a flat wall that is positioned so as to form a front side of hindrance that is first contacted by the flow of a suspension that is to be filled in the chamber, the flat wall being positioned under an angle between 0 and 90 degrees with respect to the longitudinal flow direction in the chamber. For clarity's sake, the longitudinal flow direction in the chamber is hereby defined along the flow path between the inlet and outlet of the chamber and which is parallel to the plane of the plates. The flat wall forms in practice an effective hindrance or hazard that is first contacted by the flow of the suspension and thereby diminishes or eliminates the unwanted SS effect.

According to a further preferred embodiment of the capillary loading slide according to the invention, the flat wall at the front side of the pillar disturbers has a different angle to the longitudinal flow direction in the chamber than the flat wall at the front side of the projection disturbers. Preferably, the flat wall of the pillar disturbers and the flat wall of the projection disturbers have an angle to the longitudinal flow direction of 45 and, respectively, 90 degrees. These angles have to be construed as indications of a preferred embodiment, from which deviations by 50% are also encompassed by the invention. It has been proven that the above positioning of the respective disturbers further enhances diminishing or eliminating the unwanted SS effect.

In another preferred embodiment of the capillary loading slide according to the invention, the pillar disturbers comprise a curved wall at the back side with respect to the longitudinal flow direction in the chamber. With such a design of the two opposite walls of a pillar disturber (which can be seen as a front and a back side of the platelet) , a difference in travel distance of the flow around the platelet is created, probably causing an additional turbulence to the flow. This specific design of the pillar disturber proves to enhance the reduction of the SS effect even further. Preferably, the projection disturbers of the capillary-loading slide according to invention, are arranged under a steep angle with regard to the plane of the plate to which they are connected. Such an arrangement, i.e. an angle smaller than 90 degrees, can be construed as a barb towards the direction of flow, and proves to enhance the reduction of the SS effect as well. However, also encompassed by the invention are projection disturbers which are arranged perpendicular to the plane of the plate, because of the simplicity of production. Advantageously, at least the inlet or outlet of the chamber of the capillary loading slide according to invention, comprises a partial blockade. By blocking the inlet or outlet of the chamber, a reduced flow velocity is obtained, which results in a lowering of the SS effect.

In general it is noted that the pillar disturbers are preferably positioned under a steep angle to longitudinal flow direction in the chamber in an alternating fashion regarding the orientation of the angle, i.e. so that the flow is forced to the left side at one pillar disturber and to the right side by a next pillar disturber. Furthermore it is preferred according to the invention that the pillar disturbers and projection disturbers are consecutively positioned in regard of the longitudinal flow of the chamber. An example of the positioning of the respective disturbers is provided herein below. Preferably, the capillary loading slide according to the invention comprises laminar flow disturbers which are provided within the chamber before the measuring zone. By before is meant that the laminar flow disturbers are displaced at such a position that when filling the chamber with a suspension to be measured, the laminar flow disturbers are upstream of the measuring zone, i.e. the suspension passes the laminar flow disturbers, before it reaches the measuring zone. By positioning the laminar flow disturbers as such, the goal of the invention to diminish laminar flow effects is effectively reached. Alternatively, laminar flow disturbers are displaced after, or downstream of, the measuring zone in order to diminish the unwanted effects of laminar flow. Advantageously, the laminar flow disturbers are provided both before and after the measuring zone, to further enhance the diminishing of the unwanted laminar flow effects. According to another preferred embodiment of the capillary loading slide according to the invention, the laminar flow disturbers are discontinuous recesses, e.g. small pits or lines perpendicular to the direction of the flow, provided in the inner surface of at least one of the bottom plate and top plate. These pits or lines are present in the non-measuring zones, at least in the areas between the inlet and the measuring zone. These pits or lines can be easily etched by ultraviolet lasers into the plates, which are usually manufactured from glass. The closer and the more pits or lines are present, the beter the disturbing effect on the establishment of a laminar flow.

According to an other preferred embodiment of the capillary loading slide according to the invention, the laminar flow disturbers comprise a continuous recessed zone provided in at least one of the plates. Recesses are hereby defined as cut-aways in the surface of a plate, so that the chamber height at the position of a recess is altered, c.q. enlarged. As a consequence, by enlarging the chamber height, the laminar flow in a capillary loading slide is disturbed, because a laminar flow can only develop when the spaced apart top and bottom plate are within a capillary range of distance. The distance required to obtain a laminar flow in the chamber is - amongst others - dependent of the density, and the viscosity of the suspension. It has been found that by deepening the bottom plate before the measuring zone, an effective reduction of the establishment of a laminar flow is reached. Advantageously, the recesses form together one continuous recessed zone outside the measuring zone. Typically, the recessed zone is obtained by sand blasting the surface of a bottom plate in an area or zone outside the measuring zone until a chamber height is obtained in the range of 150-200 micrometer. The recessed zone can also be realized with other methods like laser etching or etching with corrosive chemicals.

In a second aspect, the invention relates to a method of microscopic research of a suspension of particulates, in particular the counting of cell numbers of biological material in a liquid, wherein a capillary loading slide according to the invention is used and a suspension of particulates is introduced in the chamber via the inlet, followed by inspecting the suspension of particulates introduced in the chamber using a microscope. As apparent from the above, such a method of microscopic research combines the known advantage of capillary loading slide regarding the chamber height as well as the convenience of the pre-formed slide itself, with an accuracy of measurement that equals the golden standard of the hemocytometer. As a result, the measurement can be performed without need of a computation using a correction factor, while having an ideal dimensioning of the chamber in regard of chamber height. In a preferred method according to the invention the flow velocity of the suspension while being introduced into the chamber is reduced. By reducing the flow velocity of the incoming suspension, the flow velocity of the suspension in the chamber is, by consequence reduced, so that the SS effect is further suppressed. This can, for instance, be done by providing a partial blockade in the inlet. In another preferred method according to the invention the flow velocity of air that is purged out of the chamber while the suspension to be measured is introduced, is reduced. By reducing the flow velocity of the outgoing air, the flow velocity of the suspension in the chamber is, by consequence reduced, so that the SS effect is further suppressed. This can, for instance, be done by providing a partial blockade in the outlet.

To further illustrate the invention, several embodiments of the capillary loading slide are presented, both in the form of drawings and examples, and are presented hereinbelow.

Therein:

Fig. 1 shows a preferred embodiment of a capillary loading slide according to the invention in cross sectional top view.

Fig. 2A shows in detail a transverse cross section of a preferred embodiment of a capillary loading slide according to the invention.

Fig. 2B shows in detail a longitudinal cross section of a preferred embodiment of a capillary loading slide according to the invention. Fig. 3 shows in detail some laminar flow disturbers of a preferred embodiment of a capillary loading slide according to the invention, in cross sectional top view.

Fig. 4 shows a longitudinal cross section of a capillary loading slide without laminar flow disturbers, depicting the Poiseuille flow.

Fig. 5 shows a longitudinal cross section of a capillary loading slide having a recessed zone, according to another preferred embodiment.

In fig. 1, a capillary loading slide 1 is depicted in a cross sectional top view, so that a top and bottom plate are not shown here. These plates are joined to each other by a connecting layer 4, disposed between said plates. Within the boundary of the layer 4 a capillary chamber 6 is present, the chamber having an inlet 8 for introducing a suspension to be measured and an outlet 10 for purging air when the suspension is introduced. Within the chamber laminar flow disturbers are provided in the form of pillar disturbers 12 and projection disturbers 14 at fixed positions. Furthermore, both the inlet 8 as well as the outlet 10, are provided with respective blockades 16 and 18, in the form of a resin barrier (inlet and outlet) or a coarse meshed gauze or a relatively large projection disturber. At the inlet, the blockade 16 should allow the passing of particles of the suspension, whereas such is not required of the blockade 18 at the outlet 10. Also, the ideal measuring zone 20 for performing microscopic measurements is depicted. Furthermore, at the right hand side the longitudinal dimensioning of the slide 1 is noted in mm.

In fig. 2A, a bottom plate 22 and a top plate 24 are shown in transverse cross section of a detail of a capillary loading slide 1, to which a pillar disturber 12 and two projection disturbers 14 are connected. The chamber height 26 is about 20 micrometer. It is noted that the scale in horizontal direction is different than the scale in vertical direction, as is apparent from the 0,5 mm width in horizontal direction depicted at the bottom of the drawing. The pillar disturber 12 is connected to both plates 22 and 24. In this embodiment the projection disturbers 14 are fixed to one of the plates 22 and 24 in alternating manner. Furthermore additional flow disturbing elements 25 being recesses having the shape of line 25a and pit 25b (see fig. 2B) are provided in the top plate 24 and bottom plate 22 respectively.

In fig. 2B, the same features as depicted in fig. 2A, are now shown in longitudinal cross section. The direction of flow of the suspension, or the longitudinal flow direction in the chamber is indicated by arrows 30. Also, the steep angle under which the projection disturbers 14 are connected to the plates is apparent.

In fig. 3, two pillar disturbers 12 and two projection disturbers 14 are shown in cross sectional top view. The main direction of flow of the suspension is depicted by arrow 30. The actual bends that the flow of the suspension is forced into around a pillar disturber 12 are illustrated by arrows 31. The finlet-like projection disturbers 14 have flat walls 32 at their front and back sides, whereas the pillar disturbers 12 have a flat wall 34 at their front side and a curved wall 36 at their back side. In fig. 4, the Poiseuille flow is depicted by several arrows

30, in a chamber 6 between the bottom plate 22 and top plate 24, of a capillary loading slide 1 which has no laminar flow disturbers. The difference in flow velocity is dependent of the proximity to the top and bottom plates 24 and 22, as depicted by the length of the arrows 30, which lengths are commensurate to the magnitude of the flow velocity. A gradient of flow velocity over the chamber height 26 is thus apparent. In fig. 5, a longitudinal cross section of a capillary loading slide having a recessed zone 40, is depicted. The areas outside the measuring zone are sand blasted resulting in an enlargement 42 of the chamber height 20 by e.g. an additional 150 micrometer. The analysis table 44, above which the measuring zone 20 is defined, is flat and not sand blasted. No projection or pillar disturbers are provided in this embodiment. Optionally, the measuring zone 30 can comprise a grid close to the upstream rim of table 44.

Examples

Two versions of a capillary loading slide were produced according to the following description:

A slide was produced that needs no SS effect correction factor and can be used for the whole range of viscosities of bodily fluids like seminal and blood plasma. The slide according to the invention has a chamber height of 20 micrometer, projection and pillar disturbers outside the measuring area and inlet and outlet barriers A chamber height of 20 micrometer is used as it allows the use of a 1Ox microscope objective lens without loss of focus. The measuring zone of the chamber is disposed at a distance from the entrance port of about 8 mm, although other distances are convenient for measuring as well. The measuring zone is an area with a width of up to 2 mm that is used for the assessment of cell or particle number. Microscopic measurements prove that the SS effect does not develop in the measuring zone, and the concentration of cells or particles equals the true concentration, so that no correction for the SS effect is required.

A second chamber was produced that needs no SS effect correction factor and can be used for the whole range of viscosities of bodily fluids like seminal and blood plasma. This second chamber does not comprise pillar or projection disturbers but is provided with a sand blasted surface of the bottom plate outside the measuring area. The resulting chamber height is enlarged by 150 micrometer. The enlarged chamber height outside the measuring zone prevented the development of the SS effect.

For comparison, several capillary loading slides were produced bearing various features of the slide according to the invention. These slides are outlined below and the results obtained in microscopic measurements are summarized in Table 1. The slides are characterized as "chambers" having different numbers.

Chamber 1 contains one sided projection disturbers, but no pillar disturbers. Prevention of the development of laminar flow was at one side of the capillary.

Chamber 2 contains projection disturbers at both the upper and lower glass plates, but no pillar disturbers. As a consequence, the number of projection disturbers was doubled compared to chamber 1. Chamber 3 contains pillar disturbers, but no projection disturbers.

Chamber 4 contains pits or etched lines disturbers.

Chamber 5 contains an inlet barrier only.

Chamber 6 contains an outlet barrier only.

Chamber 7 contains an inlet and an outlet barrier only. Chamber 8 contains an inlet barrier, outlet barrier, pillar disturbers and one sided projection disturbers.

Chamber 9 contains an inlet barrier, outlet barrier, pillar disturbers and projection disturbers at both sides of the capillary.

Chamber 10 contains an inlet barrier, outlet barrier, pillar disturbers, projection disturbers and pit or etched lines disturbers at both sides of the capillary.

Chamber 11 contains a recessed zone with a chamber height of 200 micrometers with exception of the analysis area which has a diameter of 20 micrometers.

Experimental verification of the SS correction factor in the various designs of the capillary loading slide.

Diluted (1:40) boar semen was supplied by the Bond KI station in

Bunnik, Netherlands. The concentration was assessed at 8-8.5 mm from the entrance port with an IVOS CASA system according standard procedures.

As control a Leja chamber without flow disturbers was used. This chamber is calibrated with the hemocytometer. By multiplying the results of the standard Leja chamber with 1.30, the SS correction factor, the true concentration is known.

Each assessment was performed 25 times with the same semen sample and the experimental procedure was repeated 5 times. To make the result comparable the outcome of the control chamber was defined as 100. The outcome of the experimental chambers was divided by the outcome of the control chamber and multiplied by 100. The above chambers 1-11 were tested.

The time needed to fill chambers 1, 2 and 3 was only slightly larger than the filling time of the control chambers (2.35 seconds/about a half second longer) . Chambers , 5, 6, 7 8 and 9 needed much more time to fill. Chamber 9 needed the longest filling time, about 5 seconds

The results are depicted in table 1. For assessment of the need to correct the measurement it is explained that a correction factor is not needed when the ratio to control chamber has a value around 130. The difference to control is a value that indicates the statistic variation of the measurements.

Table 1

From table 1 it can be concluded that each experimental chamber affects the outcome. However, the efficiency differs. Chamber 3, a chamber with a pillar flow disturber, gives only an improvement of 3%. The projection flow disturbers present at both sides gives an improvement of 14%. Only the combination of inlet barrier, outlet barrier and both types of flow disturbers reaches the theoretical value of 1.30. Chamber 9, the capillary with different capillary diameters, reaches also the theoretical value of 1.30.

It is concluded that the principle of prevention of the establishment of laminar flow by flow disturbers or by increased capillary diameter in the non analysis area allow the construction of a capillary analysis chamber which does not need correction for the SS effect.

Another approach to dealing with the SS effect is to determine the SS-effect compensation factor.

According to a further aspect of the invention a method of determining an SS-effect compensation factor in a capillary loading slide is provided. This method comprises the steps of introducing a suspension of particulates into the inlet of the slide, measuring a flow characteristic of the suspension, establishing the actual number of particulates in a measuring zone, and generating an SS-effect compensation factor from correlating the filling time to said actual number.

The SS-effect depends on many factors, like chamber height, diameter of the particles, distance between inlet and place of assessment, angle of contact, surface tension, diameter of the particles or cells in the suspension, viscosity and flow velocity. Using one type of chamber and using bodily fluids all these variables are constant with the exception of the viscosity and flow velocity. The flow velocity and the viscosity are fully correlated.

By measuring a flow characteristic, preferably the filling time or flow velocity of standard capillary-loading slides (comprising a spaced apart top plate and bottom plate, said plates being joined to each other by a connecting layer disposed between said plates, wherein the layer and the plates together form a boundary surrounding a chamber for microscopic measurements, the chamber having an inlet for introducing a suspension to be measured and an outlet for purging air when the suspension is introduced, as well as a measuring zone for performing microscopic research) , and by assessing cell numbers in these slides and in the hemocytometer a relationship between filling time and SS-effect compensation factor could be constructed. The results are depicted in table 2. Table 2

The factor thus calculated can be used in processing data from microscopic research of a suspension of particulates using similar slides .

In daily practice the assessments of filling time or flow velocity of standard slides can be a burden. In view thereof the invention also provides a device for determining an SS-effect compensation factor of a capillary loading slide. This device comprises a carrying surface for holding the capillary slide, and measurement means for determining a flow characteristic of a suspension of particulates introduced in a capillary loading slide, and calculating means for calculating an SS- effect compensation factor by correlating the measured flow characteristic to the actual number of particulates in the measuring zone of the slide.

According to the invention a separate filling station is provided, in which a flow characteristic preferably the flow velocity, or filling time, is measured. This filling station can be connected to a CASA system or can be used as a stand alone. It calculates the value of the SS-effect compensation factor and the user can calculate the true concentration using standard, not corrected, slides by applying the SS-effect compensation factor thus obtained.

In a preferred embodiment of the device the measurement means comprise at least one source of light for emitting light and at least two light sensitive elements for receiving light, said elements being spaced apart in the direction of flow in the slide, said source and elements being arranged such that during operation light passes from the source through the slide to the elements.

In another preferred embodiment the device comprises temperature controlling means for heating the carrying surface. Figure 6 shows a top view of an embodiment of a device according to the invention; and Figure 7 shows a cross-section through Fig. 6 taken along the lines A-A in figure 6.

More particularly, figures 6-8 show a device 100 for determining the flow velocity in a capillary loading-slide. The device 100 comprises a recessed stage 102 for holding the slide. This stage 102 is preferably temperature controlled, e.g. by a thermostatic controller including a heating element 104. A lamp holder 106 is attached to the stage 102, and carries at its top a light source such as a light emitting diode 108. Light emitted by the diode 108 passes through a lens 110 and is focused on the slide (not shown) in two positions and received by light sensitive diodes 112 in the upper surface of stage 102. These diodes 112 are spaced apart at a known distance in the direction of flow (indicated by an arrow) in the slide to be tested. The diodes 112 respond to a distortion of the light intensity received. This distortion is caused when the front of the suspension passes the respective positions of the diodes 112. The difference in time of these responses is then calculated, and using the known distance the flow velocity can be derived. The flow velocity is correlated to the actual number of counts using a standard meter, e.g. the hemocytometer, thereby generating the SS- effect"compensation factor. Suitable connections to a power source and a computer or data storage means are also provided.

Claims

C L A I M S

1. Capillary loading slide for microscopic research of a suspension of particulates, comprising a spaced apart top plate and bottom plate, said plates being joined to each other by a connecting layer disposed between said plates, wherein the layer and the plates together form a boundary surrounding a chamber for microscopic measurements, the chamber having an inlet for introducing a suspension to be measured and an outlet for purging air when the suspension is introduced, as well as a measuring zone for performing microscopic research, characterized in that laminar flow disturbers (12,14, 25a, 25b, 40) are provided within the chamber (6) at fixed positions outside the measuring zone (20) .

2. Capillary loading slide according to claim 1, characterized in that the laminar flow disturbers (12,14) are at least connected to the top plate (24) or the bottom plate (22) .

3. Capillary loading slide according to claim 1 or 2, characterized in that the laminar flow disturbers (12,14) comprise pillar disturbers (12) that are connected to both the top plate (24) and the bottom plate (22) .

4. Capillary loading slide according to any of the preceding claims 1-3, characterized in that the laminar flow disturbers (12,14) comprise projection disturbers (14) that are connected to the top plate (24) or the bottom plate (22) and which have a height which is less than the height of the chamber between top plate (24) and bottom plate (22) .

5. ,Xap.illary loading slide according to any of the preceding claims 1-4, characterized in that the laminar flow disturbers (12,14) are made of a resin.

6. Capillary loading slide according to any of the preceding claims 1-5, characterized in that the laminar flow disturbers (12,14) comprise a flat wall (32,34) that is positioned so as to form a front side of hindrance that is first contacted by the flow of a suspension (30) that is to be filled in the chamber, the flat wall (32,34) being positioned under an angle between 0 and 90 degrees with respect to the longitudinal flow direction in the chamber (30) .

7. Capillary loading slide according to any of the preceding claims 4-6, characterized in that the flat wall (32) at the front side of the pillar disturbers (12) has a different angle to the longitudinal flow direction in the chamber than the flat wall (34) at the front side of the projection disturbers (14) .

8. Capillary loading slide according to any of the preceding claims 3-7, characterized in that the pillar disturbers (12) comprise a curved wall (36) at the back side with respect to the longitudinal flow direction in the chamber (30) .

9. Capillary loading slide according to any of the preceding claims 4-8, characterized in that the projection disturbers (14) are arranged under a steep angle, with regard to the plane of the plate (22,24) to which they are connected.

10. Capillary loading slide according to any of the preceding claims 1-9, characterized in that at least the inlet (8) or outlet (10) of the chamber (6) comprises a partial blockade (16,18) .

11. Capillary loading slide according to any of the preceding claims 1-9, characterized in that the laminar flow disturbers (12,14,25a, 25b, 40) are provided within the chamber before the measuring zone (20) .

12. Capillary loading slide according to any of the preceding claims 1-9, characterized in that the laminar flow disturbers comprise recesses (25a, 25b, 40) .

13. Method of microscopic research of a suspension of particulates, in particular the counting of cell numbers of biological material in a liquid, wherein a capillary loading slide (1) according to any of above claims 1-12 is used and a suspension of particulates is introduced in the chamber (6) via the inlet (8) , followed by inspecting the suspension of particulates introduced in the chamber (6) using a microscope.

14. Method according to claim 13, wherein the flow velocity of the suspension while being introduced into the chamber (6) is reduced.

15. Method according to claim 13 or 14, wherein the flow velocity of air that is purged out of the chamber (6) while the suspension to be measured is introduced, is reduced.

16. Method of determining an SS-effect compensation factor of a capillary loading slide, said method comprising the steps of introducing a suspension of particulates into the inlet of the slide, measuring a flow characteristic of the suspension, establishing the actual number of particulates in a measuring zone, and generating an SS-effect compensation factor from correlating the filling time to said actual number.

17. Device for determining an SS-effect compensation factor of a capillary loading slide, said device comprising a carrying surface (102) for holding the capillary loading slide, and measurement means for determining a flow characteristic of a suspension of particulates introduced in a capillary loading slide, and calculating means for calculating an SS-effect compensation factor by correlating the measured flow characteristic to the actual number of particulates in the measuring zone of the slide.

18. Device according to claim wherein said measurement means comprise at least one source (108) of light for emitting light and at least two light sensitive elements (112) for receiving light, said elements being spaced apart in the direction of flow in the slide, said- source and elements being arranged that during operation light passes from the source (108) through the slide to the elements (112) . \

19. -Device according to claim 17 or 18, comprising temperature controlling means (104) for heating the carrying surface (102) .