DE102007019230A1 - Sample vessel for centrifugation and packed cell volume measurement of e.g. blood cell suspensions, includes surfaces for light introduction into capillary - Google Patents

Abstract

One or more surfaces are provided in the sample tube (10) to allow light to be coupled-into the wall of the capillary (11) and/or the holding region. At the closed end of the capillary (11) including the measurement region (II), a surface is provided for light introduction. There are one or more such surfaces on the inner wall of the capillary, which has an internal diameter of about 500 mu m. The capillary preferably has a volume of about 5 mu l. The respective cross sections are constant with height. The wall is transparent, plastic and preferably made of polycarbonate. Stabilizing reinforcements (12), e.g. fins, are molded onto the side of the capillary, over the length of the measurement region. They orientate the tube, to provide a perpendicular line of sight to the measurement region. The light coupling surfaces are close to the reinforcements.

Description

The The invention relates to a sample vessel for centrifuging a Cell suspension with a measuring range in which after centrifugation Solid material present in the sample as a compacted cell cake Deposits, and with a receiving area, in which after centrifugation liquid supernatant collects, with the measuring range as at least sectorally transparent Capillary is formed whose clear width much lower is the clear width of the reception area.

Out The area of medical diagnostics is the so-called PCV procedure known. PCV stands for packed cell volume "and describes the proportion of solids in the form of a compacted cell cake on the total volume of a sample. Widely known as the hematocrit designated PCV value of the blood. To its determination becomes one Blood sample centrifuged in a mostly cylindrical sample tube until the solid component of the blood as a compacted cell cake on the bottom of the tube has dropped off. The as a supernatant designated liquid fraction Floats on the cell cake. For determination of hematocrit the volume of the cell cake is determined and expressed in a percentage relationship set to the total volume of the sample.

typical Measurements resulting from the hematocrit measurement procedure described above move in the range of 30-50%. For typical cell cultures, the expected total solids values are significantly lower, typically in the range of 1% or less. This means that when using the usual sample vessels for PCV measurements on such cell cultures, the height of the supernatant column about one hundred times the height of the cell cake. This can easily be too big Lead to read errors.

It are here as PCV tubes designated sample vessels known which eliminate this disadvantage in that each sample vessel a Recording area and a subsequent to this, as Capillary trained measuring range, whose clear width is much smaller than the clear width of the receiving area. This will be a scale transformation achieved so that a given volume of a much greater height in the measuring range as in the recording area corresponds. This means a relative scale spread in the area of the cell cake, so that a reading error, i. a wrong one Determination of height of the cell cake, only a small error of volume determination leads. On the other hand lead these PCV tubes too to a relative scaling in the recording area. That means, that the absolute error introduced by a reading error in the volume determination of the supernatant while larger as the absolute error of the cell cake volume determination; by virtue of of very large supernatant total volume However, the crucial, relative error remains acceptable In particular, it is not larger than when using otherwise usual Sample vessels.

to optical detection of heights of cell cake and / or supernatant appropriate illumination of the sample vessel is required. The characteristics hang the lighting in particular of the chosen Detection mode, i. Transmission, reflection, scattering, fluorescence etc. from. A common one Detection mode is the reflection. However, this is disadvantageous there are reflexes of the tube wall that of the cell cake or the supernatant superimpose reflected light, which in particular in automated detection errors to lead can.

It It is the object of the present invention to provide a known PCV tube in such a way to further develop that improved ascertainability of the heights of Cell cake and / or supernatant guaranteed is.

These Task is combined with the features of the preamble of Claim 1 solved by at least one light input surface is provided over which Light in the wall of the capillary and / or the receiving area can be coupled. It is preferred that at the closed end of the measuring range comprehensive capillary provided a light input surface is. This can e.g. as a transparent flattening of the capillary tip be trained, leading to the surfaces the Kapillarenwandung is at such an angle that in the coupling surface induced light by total reflection in the capillary wall propagates. The sample vessel itself or its wall thus serves as a light guide.

Of the Refractive index of the wall material is preferably to be chosen so that towards the outside of the vessel the total reflection as possible is perfectly realized while to the vessel interior, i.e. to the cell cake or to the supernatant out, lights are decoupled. This decoupled light is from the cell cake or the supernatant Characteristically scattered and penetrates the transparent vessel wall to there to be detected by the eye or a detector.

The decoupling of the light into the vessel interior can take place at particular provided outcoupling points or along certain coupling-out lines, which are distinguished, for example, by roughening their surface and / or special coupling-out prisms. Such decoupling surfaces are preferred To arrange outside the observed wall area of the capillary so as not to hinder the measurement. Often, however, even the lower refractive index difference between capillary wall and cell cake / supernatant in comparison to the ambient air will be sufficient to ensure efficient decoupling.

Preferably is provided in the sample vessel according to the invention, that its wall of a transparent material, in particular a transparent plastic, preferably of a transparent Polycarbonate exists.

The Transparency is important so that an optical detection of the height of the cell cake or the supernatant column is made possible. The terms "optical" and "transparent" are not on the frequent designated as the optical spectral range between about 400 and 800 nanometers wavelength limited to the electromagnetic spectrum. Especially when using of dyes to improve the contrast between cell cake and supernatant and / or between different areas of the cell cake may also Light of the infrared or ultraviolet spectrum for detection be used. The terms "optical" and "transparent" are then correspondingly interpret.

When The use of a capillary has proven to be particularly suitable for practical use with an inner diameter in the range of 500 microns and a Volume from one to ten microliters, in particular two to seven microliters, and more preferably about five microliters proven as a measuring range. The receiving area, its diameter in the order of magnitude from a few millimeters to a few centimeters preferably a volume of the order of milliliters, in particular two milliliters or less, preferably about one milliliter, on. This dimensioning leads that in the investigation of typical cell cultures compacted cell cake forms only in the measuring range, whereas the receiving area after centrifugation exclusively with supernatant filled is. If not complete filled with cell cake Measuring range is additional to the receiving area also the upper part of the measuring range with overhang filled be. However, this volume is easy knowing the geometry of the sample vessel according to the invention determinable and in many cases in the result even negligible be.

The Volume determination is preferably carried out by determining the heights of Cell cake or the supernatant, from those with knowledge of the geometry of the sample vessel the corresponding volume can be determined. This is especially easy if the measuring range and preferably also the receiving area, as in a preferred embodiment provided an over their height have substantially constant cross-section. The height distribution The individual sections then correspond directly to the volume distribution. To achieve this advantage, it is sufficient if the consistent cross sections over each extending the essential part of the measuring or recording area. Changes in cross section in transition between the recording area and the measuring area and / or at the lower end of the measuring range do not detract, because these areas always with supernatant or cell cake filled be and thus only a constant, additive contribution to each by height measurement deliver determined volumes.

As explains is the capillary forming the measuring area a very filigree structure, correspondingly easily damaged can be. To counter this, is in a training the invention provides that the side of the measuring range or a several stabilizing bodies are formed. These are preferably integral with the capillary and formed the receiving area. However, the stabilizer or bodies must always be such that in at least one orientation of the sample vessel one vertical line of sight on the measuring range and preferably also is undisputed on the recording area. This is preferably done in that a plurality of stabilizing wings are provided as the stabilizing body, which are longitudinal extend the capillary axis. Preferably, exactly two become each other opposite stabilizing fins however, other numbers of stabilizer blades are used are feasible. Alternatively, as a stabilizing body also a sectorwise, axial continuation of the cylindrical shaped receiving area be provided.

Further Features and advantages of the invention will become apparent from the following, special description as well as the drawings.

It demonstrate:

1 a schematic representation of a preferred embodiment of a sample vessel;

2 a view rotated by 90 ° about the axis of symmetry of a sample vessel according to 1 ;

3 : an enlarged view of the measuring range of a sample vessel according to 1 ;

4 : An enlarged view of the closed end of the measuring range to illustrate the preferred light coupling.

1 and 2 show a schematic representation of a particularly preferred embodiment of a sample vessel, namely a so-called PCV tube 10 , The PCV tube 10 can be divided into two main areas. A first, large-volume area I is referred to here as receiving area I. A second area II designed as a capillary is referred to here as a measuring area. The recording area is at the in 1 and 2 illustrated embodiment divided into two sub-areas: a substantially cylindrical main area Ia and a tapered transition area Ib. Typical dimensions for the receiving area I are in the order of centimeters, its typical volume in the order of about 1 ml. The measuring area II is formed as a capillary with a typical clear width of about 500 microns. Its typical volume is about 5 μl. It should be expressly noted that these measures are only examples of good practice in practice. These may, without departing from the gist of the present invention, be adapted by those skilled in the art in light of the specific application. Especially in cases where high PCV values are to be expected, the volume of measuring range II can be made larger. In cases where cultures of particularly large cells are to be examined, for example, the inside diameter of the capillary of the measuring range II can be made correspondingly large. The opposite, of course, applies analogously to cases of particularly small cells and / or lower expected PCV values.

The PCV tube is at least in its measuring range II of transparent material, preferably made of transparent plastic, in particular made of a transparent Polycabonat. At the in 1 and 2 shown, particularly advantageous embodiment are adjacent to the measuring range II and the transition region Ib two stabilizing wings 12 intended. The stabilizing wings 12 are preferably formed integrally with the outer walls of the measuring region II and the transition region Ib and run such that, at least for a given orientation of the PCV tube 10 an unobstructed view of the measuring range II is possible. Although at the in the 1 and 2 illustrated embodiment, two stabilizing wings 12 are shown, their number is not limited in principle. Also it could be in place of wings 12 to act one or more stabilizing body, for example in a cylindrical continuation of the main receiving area Ia. However, what is important is the unobstructed ability to see in at least one orientation of the PCV tube 10 ,

To carry out a PCV determination, an optionally suitably stained cell suspension in the PCV tube is first prepared 10 centrifuged. Centrifugation at about 2500 g over about 1 min has proven particularly favorable. Significantly lower relative centrifugal forces and / or shorter centrifugation times lead to insufficient compaction of the cell cake. In contrast, re-expansion of the cell cake after completion of the centrifugation step is observed with significantly larger relative centrifugal forces. This leads to unwanted measurement inaccuracies. Significantly longer centrifugation times lead to no appreciable compression improvement or reproducibility of the result and are therefore not meaningful due to the directly associated with extending the duration of the procedure. It should be noted, however, that particular cell types require other centrifugation parameters to optimize results. Making an appropriate choice is within the ordinary skill of the art.

3 shows the measuring range II of a PCV tube 10 in an enlarged, schematic representation. In the lower part of the capillary has a cell cake during centrifugation 13 discontinued. The cell cake 13 is divided into three sections of different heights. In a first cell cake section 131 due to the high density of the material forming it in the lower part of the capillary 11 settles, are the completely intact cells. In another area 133 due to the particularly low density of the material forming it, the uppermost portion of the cell cake 13 forms, dead cells and cell fragments are included. Between the areas 131 and 133 extends an area 132 containing the cells whose degree of viability is completely intact and completely dead. The various areas can be visually distinguished with a suitable staining of the cell suspension with a so-called. Viabilitätsfarbstoff. Only the scale spread in the measuring range as a result of the sample vessel according to the invention also makes it possible to measure these differences.

There this is z.T. is small optical differences is a very good lighting appropriate. As previously explained These are done by simply lighting the sample vessel with illumination light. However, this carries the risk of unwanted reflections on the outer vessel wall.

4 shows a sample container according to the invention, which allows a particularly advantageous type of lighting. Shown is the lower part of the capillary 11 , which forms the measuring range II. Schematically shown are the capillary wall 110 with an outer surface 111 and an inner surface 112 , The capillary wall 112 encloses the interior 114 who with the in 4 not shown cell cake ge is filled. The interior 114 runs in the illustrated embodiment in a tip, which is mainly due to manufacturing technology. The outside of the capillary wall 110 However, it is flattened in the area of the tip and forms a light coupling surface 116 through the light 202 a light source 200 into the capillary wall 110 can penetrate. With a correct choice of refractive index and coupling angle, which the skilled person can deduce based on known optical laws, experiences the injected light 202 Total reflection. The total reflection is on the outer surface 111 the capillary wall due to the large refractive index difference to the surrounding air almost perfect. On the inner surface 112 however, the capillary wall, which is the interface to the cell cake that is optically more dense than the ambient air, becomes part of the light 202 in the interior 114 the capillary decoupled. This corresponds to a lighting of the cell cake. The decoupled light 204 is scattered and / or at least partially absorbed in the cell cake and sent out through the capillary wall at angles which do not produce total internal reflection (in FIG 4 not shown) where it can be detected by eye or a suitable photodetector. In the embodiment shown thus serves the capillary wall 110 as a light guide for the illumination light illuminating the cell cake perpendicular to its height.

This is especially when stained Cell cake advantageous because a lighting along the cell cake axis because of the strong absorption to different illuminances in would lead to the different cell cake sections. The lighting vertical on the other hand, to the cell cake axis through the capillary wall the above-mentioned reflection disadvantages.

alternative or additionally is also a corresponding lighting from above possible, the annular upper edge of the receiving area I at suitably flat trained edge surface as a coupling surface can serve.

Of course, ask those discussed in the specific description and in the figures shown embodiments only illustrative embodiments The expert is a wide range at modification possibilities to disposal.

Claims (11)

Sample vessel for centrifuging a cell suspension having a measuring range (II), in which, after centrifugation in the sample, solid material present as a compacted cell cake ( 13 ) and with a receiving region (I), in which liquid supernatant collects after centrifugation, the measuring region (II) being at least sector-wise transparent capillaries (II). 11 ) whose inside width is substantially smaller than the clear width of the receiving region (I), characterized in that at least one light coupling surface is provided, via which light into the wall of the capillary ( 11 ) and / or the receiving area can be coupled. Sample vessel according to claim 1, characterized in that at the closed end of the measuring range (II) comprehensive capillary ( 11 ) a light input surface ( 116 ) is provided. Sample vessel according to one of the preceding claims, characterized in that on the inner wall ( 112 ) of the capillary ( 11 ) One or more Lichtauskoppelflächen are provided. Sample vessel according to one of the preceding claims, characterized in that the capillary ( 11 ) has an inner diameter of about 500 microns. Sample vessel according to one of the preceding claims, characterized in that the capillary ( 11 ) has a volume of 1 to 10 microliters, in particular 2 to 7 microliters, in particular of about 5 microliters. Sample container after one of the preceding claims, characterized in that the measuring area (II) and / or the receiving area (I) one each over their height have substantially constant cross-section. Sample vessel according to one of the preceding claims, characterized in that its wall ( 110 ) consists of a transparent material, in particular a transparent plastic, in particular of a transparent polycarbonate. Sample vessel according to one of the preceding claims, characterized in that laterally to the capillary (11) comprising the measuring region (II) ( 11 ) one or more stabilizing bodies ( 12 ) are formed. Sample vessel according to claim 8, characterized in that a plurality of stabilizing wings ( 12 ) are provided. Sample vessel according to one of claims 8 or 9, characterized in that the stabilizing body or bodies ( 12 ) are arranged so that in at least one orientation of the sample vessel a vertical line of sight on the measuring range is undisputed. Sample container after one the claims 4 to 10, as far back to claim 2, characterized in that the light output surfaces in Area of stabilization body are located.