COUNTING COMPARTMENT FOR BIOLOGICAL INVESTIGATIONS AND A METHOD FOR MANUFACTURING SUCH A COUNTING COMPARTMENT

Abstract: Counting compartment (1) having an inlet (5) and an outlet (6), and comprising a bottom plate (2) and a top plate (3), which are held at a distance from each other by material particles which have the same size and are situated at a distance from one another. The particles are surrounded by and embedded in a solid connecting layer formed by an adhesive. The connecting layer extends all round and up to the free end (11) of the outlet of the counting compartment. A fluent mixture of an adhesive mixed with electrostatically charged particles is used to form the counting compartment (1), the largest size of the material particles determining the depth of the counting compartment. The counting compartment is particularly suitable for counting cells in biological materials such as sperm and blood samples.

Description: Title: Counting compartment for biological investigations and a method for manufacturing such a counting compartment The invention relates to a counting compartment for accommodating a sample of biological material consisting of microscopic particles in a liquid medium for the quantitative microscopic examination of this material. When examining body fluids, it is often desirable to study samples of such biological material under the micro¬ scope over a certain time. When examining sperm, for example, the aim is to establish how many spermatozoa are present in the sample and also how motile they are. To carry out this investigation, a sample of a certain thickness is to be subjected to microscopic examin¬ ation in a counting compartment, using a grid built into the eyelens of the microscope. Such a grid may be divided into a hundred squares, and the number of spermatozoa in each of a representative number of squares can be counted by the investigator in order to determine the total number of spermatozoa in the whole grid area. Such a grid may also be provided in the counting compartment. The number of spermato¬ zoa in a square can be e.g. about one hundred to twohundred. From US-A- 4 911 782 and 5 200 152 a method is known for conducting such determinations with the aid of a counting compartment formed by two transparent plates joined together by a connecting layer composed of a cured plastic. However, the manufacture of such a counting compart- ment is complicated and expensive, because it involves the use of a photo-resist method for applying a plastic as the desired boundary of the compartment. The aim of the present invention is to provide a counting compartment and a manufacturing method for it, which do not have this shortcoming and do not involve the use of the photo-resist method. This aim is achieved according to the present inven¬ tion by a counting compartment of the type indicated above, which is characterized in that the connecting layer contains material particles which are separate from one another and have a size which determines the depth of the counti compartment, which material particles are essentially contact with the two plates. The counting compartment can easily be constructed using material particles which have a size which determin the depth of the counting compartment; furthermore, counti compartments with depths suitable for different biologic materials can be produced by using material particles appropriate dimensions. The fact is that the examination of a sperm samp calls for a counting compartment with a depth of 12- microns, while the examination of blood cells needs a coun ing compartment with a depth of only 8-12 microns. Counti compartments with the required depth can be made by choosi material particles of the right size from amongst materi particles measuring between 6 and 40 microns. The particles which determine the depth of t counting compartment lie at a regular distance from o another in order to ensure a uniform depth for the counti compartment over its entire surface. Preferably, the particles, when their masses are equal, are at equal di tances from one another. Furthermore, and preferably, the particles are substantially completely surrounded by t solid material of the connecting layer, formed from a flue material. To prevent clotting of these material particles, t particulate material chosen is preferably a non-magneti electrically highly resistant substance and, more preferabl aluminium oxide, but other materials and even plastic pa tides can also be used. To prevent escaping, by capillary action, and evapo ation of liquid from the sample of biological materi consisting of microscopic particles in the counting compar ment, the solid material of the connecting layer extends a round and up to the free end of the outlet of the compar ment. This minimizes evaporation, so that no streaming occu in the sample housed in the counting compartment. The top and bottom plate are preferably both made a material which transmits UV and/or visible light, prefera ly glass, and the top plate should be selected to be as thin as possible in order to facilitate the examination by means of a microscope. The invention also relates to a method for the manufacturing of a counting compartment to accommodate a sample of biological material consisting of microscopic particles in a liquid medium for the quantitative microscopic examination of this material, which counting compartment comprises two transparent plates held at a fixed distance from each other and joined together by a connecting layer, and at least one counting compartment situated between the plates, bounded by the connecting layer and fitted with an inlet and an outlet. According to the invention, this method is characterized in that a fluent mixture of mutually repel- ling material particles and a liquid adhesive is applied to one of the plates, the other plate is placed on this fluent mixture, and pressure is exerted thereon until the two plates are essentially in contact with the largest material par¬ ticles in the fluent material, which largest material par- tides determine the depth of the counting compartment. It will be obvious that by using the above liquid mixture, one can easily apply this mixture to one of the plates, and the other plate can then be pressed on to this liquid material until the two plates are in contact with the largest particles present in the fluent mixture. The mutually repelling material particles preferably carry an electrostatic charge and consist of a non-magnetic, electrically strongly insulating, material and are then charged electrostatically. By using particles carrying an electrostatic charge, the particles will always lie at the same distance from one another, this distance depending on the size of the particle and on the charge. A counting compartment according to the invention can be used to carry out biological determinations on samples of sperm, of blood and of malignant tissue, such as cancer. From US-A 4 689 307 it is known to provide microbeads as spacers between microscope slide and cover-slip around a sample for microscopic investigation. They can be added in advance to an aqueous liquid. From EP-A 0 321 889 it is known to provide microbea onto a microscope slide by fixing them in advance in a sol residue of dried liquid adhesive. This adhesive is soluble the sample to be examined. Neither one of these publications discloses the use microparticles in a solid connecting layer for the formati of a counting compartment. Furthermore, there is no indic tion for the manner in which microspheres or other materi particles which determine the spacing can be added witho clotting to a viscous mass which, after curing, will n dissolve into the sample under examination. The invention will be explained in more detail bel with reference to the drawing of an illustrative embodimen in which: Fig. 1 shows a plan view of a counting compartme according to the invention; Fig. 2 shows a plan view of the bottom plate of t counting compartment after printing and the application of fluent mixture of adhesive and material particles thereto Fig. 3 shows a cross-section taken along line III-I in Fig. 1; Fig. 4 shows a cross-section taken along line IV- in Fig. 1, and Fig. 5 shows a view according to Fig. 1 but with pa of the top plate removed. The counting compartment 1 is formed by taking a fl glass bottom plate 2 and printing on one of its faces, pattern which comprises strips 12 and 13 at both ends of t plate, and two strips 14 along each of the edges of t plate. Furthermore, preferably on the face of bottomplate oriented towards top plate 3 to be provided later, a strip is printed, bounding the periphery of a counting compartmen by which the examination with such counting compartment made easier. The height of strips 12, 13, 14 and 15 is abo 4-6 microns. The inlet 5 of the compartment is formed by a spa 4 in the end strip 12 or 13 adjoining the boundary strip 1 Information 16 relating to the contents of t counting compartment can be given on the strips 12 and 13. These strips 12, 13, 14 and 15 are made by means of a doctor blade using ink or dabber printing technique. The ink used is allowed to dry in air or, depending on the type of ink used, is made to set under UV light. The ink used should, as a matter of course, adhere to the glas, and it may not be soluble in bodily fluids. A fluent mixture consisting of a polyester adhesive 9 (fig. 3) and aluminium oxide particles 8, which have previously been electrostatically charged and which are therefore mutually repellent, is then applied to the bottom plate 2, in form of strips 10 (fig. 2). The material par¬ ticles 8 having the largest dimensions will determine the depth of the counting compartment 1. A suitable maximum size of the particles can be selected beforehand by first screening the material, which also permits the depth of the counting compartment to be regulated. When strips 10 of said fluent mixture have been applied, a top plate 3 is pressed on until the bottom surface thereof contacts the largest of the material particles 8 present in the mixture (seefig. 3) . At the same time, the fluid mixture of strips 10 is spread to form a connecting layer 7, which extends at least all round and up to the free end 11 of the outlet 6 of the compartment. The connecting layer 7 is then allowed to solidify so as to obtain a solid connecting mass. Evaporation of a sample introduced into the counting compartment is minimized by ensuring that the solid connect- ing layer 7 extends all round and up to the free end 11 of the outlet of the compartment. This eliminates streaming in the sample to be examined under a microscope. It is important for the connecting layer to fill the space between bottom plate and top plate at least up to the edges of the plates, in order to oppose lateral spreading of the sample situated close to the outlet 6 by capillary effect. Expediently, the bottom plate 2 is a standard micro¬ scope slide measuring 7.5 x 2.5 cm, and the top plate is a standard cover slip measuring 3.2 x 2.5 cm. -- - 6 - As can be seen from Fig. 1, two counting compartme 1 have been formed on the bottom plate. The solid connect mass 17 separating the two counting compartments forms adequate barrier which prevents the sample material f moving from one counting compartment to the other. counting spermatozoa preferably elongated compartments a used. Counting compartments with a depth of 12, 20, 30 a 40 microns can be formed by using aluminium oxide particl 8 whose largest dimensions are 6 or 8, 10, 18, 28 and microns, respectively. When counting spermatozoa 8' in sper it is expedient to set the counting compartment at a dep obtained by using particles, the largest of which measure microns, with which the compartment depth becomes 20 micron In addition to these particles with the required maxim dimensions, the material particles 8 can of course al contain smaller particles 8, but they do not determine t distance between the bottom plate 2 and the top plate 3. Owing to the electrostatic charge applied to t aluminium oxide particles 8 which are mixed with t polyester adhesive 9, the largest particles in the finish counting compartment 1 will all be situated at the sa distance from one another (f, see Fig. 5) on account of t repulsion between these particles. Although aluminium oxide particles 8 are mention above, plastic particles can also be used, provided that th are not soluble in, attacked by or deformed by the adhesiv Glass beads can also be used, although they are somewhat mo difficult to charge on account of their hycroscopic propert Another suitable material for example, is magnesium oxid In addition to the largest particles 8 , the materi can of course also contain a number of smaller alumini oxide particles, since a fraction with the largest particl as the upper size limit is screened out of the starti material. Electrostatically, preferably positively charging t particles is preferably carried out by making the particl move through an electrostatic field. After that, the pa ticles are collected in a glass container, in which t adhesive is added, the combination being stirred until the particles are divided evenly in the adhesive. As a result of the charge, no clots will form but the particles will come to lie at regular distances, determined by mass and charge. The amount of material particles 8 added to the adhesive is suitably less than 10% and preferably at most 3%. Amounts of more than 3% do not give any further improvement. The polyester adhesive 9 can be replaced by a silicone adhesive, a metacrylic resin or other synthetic resin. In any case the requirement is that the material must not be soluble in bodily liquids. Preferably also adhesive 9 is of a type which is UV-curing.

Claims: CLAIMS 1. Counting compartment (1) for accommodating a samp of biological material consisting of microscopic particles a liquid medium for the quantitative microscopic examinati of this material, which counting compartment comprises t transparent plates (2, 3) which are held at a fixed distan from each other and joined together by a connecting lay (7) , and at least one counting compartment (1) which situated between the plates, bounded by the connecting lay (7) and fitted with an inlet (5) and an outlet (6) , chara terized in that the connecting layer (7) contains materi particles (8) which are separate from one another and have size that determines the depth of the counting compartmen which material particles are substantially in contact wi the two plates (2, 3). 2. Counting compartment according to claim 1, characte ized in that the material particles (8) which determine t depth of the counting compartment lie at regular distanc from one another. 3. Counting compartment according to claim 1, characte ized in that the material particles (8) which determine t depth of the counting compartment, when their masses a equal, lie at equal distances from one another. 4. Counting compartment according to any of claims 1- characterized in that the said material particles (8) consi of a non-magnetic, electrically higly insulating material. 5. Counting compartment according to any of the prece ing claims, characterized in that the said material particl (8) are completely surrounded by a solid connecting layer ( formed from a fluent material. 6. Counting compartment according to any of the prece ing claims, characterized in that the solid connecting lay extends all round and up to the free end (11) of the outl (6) of the compartment. 7. Counting compartment according to any of the prece ing claims, characterized in that the top plate (3) selected to be as thin as possible. 8. Counting compartment according to any of the precending claims, characterized in that on at least one surface of one of the plates a strip (15) is provided for delimiting a counting compartment. 9. Method for manufacturing a counting compartment to accommodate a sample of biological material consisting of microscopic particles in a liquid medium for the quantitative microscopic examination of this material, which counting compartment comprises two transparent plates (2, 3) held at a fixed distance from each other and joined together by a connecting layer (7) , and at least one counting compartment (1) which is situated between the plates, bounded by the connecting layer (7) and fitted with an inlet (5) and an outlet (6) , characterized in that a fluent mixture (10) of mutually repelling material particles and a liquid connecting material is applied to one of the plates (2) , the other plate (3) is placed on this fluent mass (10) , and pressure is exerted thereon until the two plates are substantially in contact with the largest particles (8) in the fluent material, which largest material particles determine the depth of the counting compartment. 10. Method according to claim 9, characterized in that the mutually repellent material particles (8) are given an electrostatic charge before being mixed with the material of the connecting mass. 11. Method according to claim 10, characterized in that the material particles (8) are particles of a non-magnetic, electrically highly insulating material. 12. Method according to any of claims 9-11, characterized in that the fluent mass is allowed to extend all round and up to the free end (11) of the outlet (6) of the compartment. 13. Method according to any of claims 9-12, characterized in that the top plate (3) is selected to be as thin as possible.

Inventors: DE KOCK ALFONS PETRUS ANTONIUS, VERMEIDEN JAN PIETER WILLEM, DE KOCK, ALFONS, PETRUS, ANTONIUS, GERRIT, VERMEIDEN, JAN, PIETER, WILLEM
Applicants: KOCK ALFONS PETRUS ANTONIUS GE, RODAAL BEHEER B V, VERMEIDEN JAN PIETER WILLEM, DE KOCK, ALFONS, PETRUS, ANTONIUS, GERRIT, RODAAL BEHEER B.V., VERMEIDEN, JAN, PIETER, WILLEM
Application year: 1996
Main classification: G02B21
IPC codes: B01L3, G01N1, G01N15, G01N33, G02B21, G06M11