Tissue processing method and tissue processing device

Abstract

Ensure proper transitions between each process are possible. A method for tissue processing of at least one biological tissue (105), comprising treating the at least one tissue with at least one fluid (110, 120, 130, 140), measuring a rate of concentration change in the at least one fluid (110, 120, 130, 140), and performing an action dependent on the measured rate of concentration change.

Description

[Technical field]

[0001] The present invention relates to a method for tissue processing of at least one biological tissue and to a tissue processing device usable therefor. [Background technology]

[0002] Tissue processing devices can be used, for example, to prepare biological tissue for which it is desired to perform a pathological examination. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] DE 10 2009 038 481 A1 [Patent Document 2] DE 10 2008 054 071 A1 Summary of the Invention [Problem to be solved by the invention]

[0004] For example, such processing devices are described in DE 10 2009 038 481 A1 or DE 10 2008 054 071 A1. [Means for solving the problem]

[0005] According to the invention there is provided a tissue processing method and a tissue processing device having the features of the independent claims. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0006] Advantageous configurations are the subject of the dependent claims as well as the following description.

[0007] In particular, the method of tissue processing of at least one biological tissue according to the invention comprises processing the at least one tissue with at least one liquid, measuring a rate of change of concentration in the at least one liquid, and performing a treatment depending on the measured rate of change of concentration. Thus, it is possible to determine the proper end of the respective treatment step (as the rate of change of concentration decreases, the tissue is less chemically altered) and to perform a corresponding treatment, for example by outputting a signal to indicate the end of the treatment step or by automatically starting a subsequent treatment step.

[0008] In particular, the treatment can include at least partially staining at least one tissue, so that excessive staining can be avoided, which has a positive effect on the detectability of the tissue features to be examined.

[0009] Alternatively or additionally, the treatment of the at least one tissue may comprise, in sequence and in a purposefully determined sequence, one or more of the following steps: fixation of the at least one tissue with at least one fixative, wherein the at least one tissue is hardened; liquid exchange with a polar, non-aqueous exchange solvent, wherein water is expelled from the at least one tissue and replaced by the exchange solvent; clarification with a clarifying liquid, in particular comprising an organic solvent less polar than the exchange solvent, wherein the exchange solvent in the at least one tissue is replaced by the clarifying liquid; and impregnation of the at least one tissue with an impregnating agent, wherein the clearing liquid in the at least one tissue is replaced by the impregnating agent; and wherein measurements of the concentration change rates in the fixative and / or the exchange solvent and / or the clearing liquid and / or the impregnating agent are performed during each use, and at least one procedure can be performed depending on at least one of the measured concentration change rates. This makes it possible to achieve a constant preparation quality for the tissue sample(s), independent of size, shape or other characteristics, and also ensures that excessive or insufficient penetration of the respective liquid into the tissue is avoided.

[0010] The actions can include, inter alia, terminating the respective method step during which a rate of change in concentration was measured and proceeding to the respective next method step if the rate of change in concentration falls below a predefinable threshold, thereby allowing a large degree of automation of the sample preparation.

[0011] For example, the concentration change rate threshold can be less than 20, 10, 5 or 2% / min [with respect to the absolute concentration of each species]. With such small change rates, no significant impact on the overall quality of the tissue processing can be expected any more. Alternatively or additionally, the threshold can be determined depending on the size, mass or volume of at least one tissue in order to guarantee constant processing conditions even with variable sample sizes. It is also advantageously possible to impose quality requirements such that a small threshold is selected when the requirements are high and a larger threshold (and therefore a shorter processing time) is selected when the requirements for quality are lower. It should be noted that doubling the threshold does not usually halve the processing time, since the observed concentration changes typically follow an exponential function.

[0012] In advantageous embodiments, the method further comprises measuring at least one absolute concentration in the liquids, in particular in the fixative and / or exchange solvent and / or clarifier and / or impregnating agent, and performing (starting) an exchange of the respective liquid when the measured absolute concentration reaches a predefinable threshold value, thereby ensuring a minimum quality of one or more fluids, so that the method can be economically optimized with regard to the use of the liquids without having to tolerate a loss of quality.

[0013] It should be emphasized here that each of the above process steps(es) can also comprise several essentially similar individual steps. For example, the liquid exchange can be carried out by several, for example 2, 3, 4, 5 or 6 or more sequential (arranged) baths in the respective exchange solvent. The same applies to the other process steps mentioned above.

[0014] Typically, the fixative contains 1-4% formaldehyde in aqueous solution, stabilized with 0.5-2% methanol to counteract autopolymerization. In addition, the fixative may contain a phosphate buffer (e.g., potassium dihydrogen phosphate and / or disodium hydrogen phosphate dihydrate 1-12 g / 1000 mL).

[0015] The exchange medium typically contains a mixture of water and simple alcohols (e.g. ethanol and / or isopropanol) with an alcohol content of 10-99.9%. During fluid or medium exchange, lipids, especially fats and sphingolipids (especially ceramides) and lipoproteins are usually eluted from the tissue. These substances may make up a total proportion of 1-15% of the exchange medium, especially towards the end of the first fluid exchange step.

[0016] The clarified liquid typically contains xylenes and / or other aromatic hydrocarbons (e.g., toluene) and simple alcohols (one or more of the alcohols also contained in the exchange solvent), with the proportion of aromatic species in the clarified liquid ranging from 10 to 99.9%.

[0017] As the impregnating agent, histological paraffin that can contain the aromatic substances contained in the clear liquid is usually used, and the proportion of paraffin in the impregnating agent is 70 to 99.9%.

[0018] All information (or data) on concentrations and ratios refer to the respective volumes of mixtures considered. Typically, these concentrations may increase as one proceeds from one bath to the next in a single processing step.

[0019] A tissue processing device according to the invention comprises at least one sensor adapted to measure the concentration and / or rate of change of concentration of a liquid used in the tissue processing device, and means for configuring the tissue processing device to perform the above-mentioned method, so that the tissue processing device accordingly benefits from the advantages described for the corresponding method, and vice versa.

[0020] The sensor is preferably configured to measure the concentration and / or the rate of change of concentration based on the speed of sound in the liquid and / or the acoustic impedance of the liquid and / or the temperature of the liquid and / or the chemical and / or electrochemical and / or electrical behavior of the liquid and / or the optical properties of the liquid, which are particularly suitable parameters from which accurate conclusions can be drawn about the concentration of the liquid or its rate of change.

[0021] Further advantages and embodiments of the invention become apparent from the description and the accompanying drawings.

[0022] It is understood that the features mentioned above, and those to be further described below, can be used not only in the combinations indicated in each case, but also in other combinations or alone, without departing from the scope of the invention.

[0023] The invention is illustrated diagrammatically in the drawings by way of an embodiment and will be explained below with reference to the drawings, in which, to avoid repetition, the drawing reference signs referring to components of an apparatus are also used to refer to method steps performed in the apparatus and vice versa. [Brief description of the drawings]

[0024] [Figure 1] 1 is a schematic diagram of an advantageous embodiment of one example of a tissue processing apparatus of the present invention; [Diagram 2] 1 shows an example of a typical concentration profile (curve) that may occur in a liquid within the scope of the present invention. EXAMPLES

[0025] 1 is a schematic diagram of one advantageous embodiment of a tissue processing apparatus according to the present invention, generally designated 100. As shown in FIG.

[0026] The tissue processing apparatus 100 includes at least one retort (container) 103 adapted and configured for receiving and filling with at least one organic liquid (fluid) one or more biological tissues or samples 105. Among other things, the retort can include one or more materials from the group consisting of stainless steel, aluminum and other metals, polyethyletherketone (PEEK), polytetrafluoroethylene (PTFE), polyethylene (PE), polypropylene (PP), polycarbonate (PC) and other suitable plastics and glass.

[0027] Furthermore, the tissue processing device 100 comprises a sensor 107 configured to measure the concentration and / or the rate of change of the concentration and / or other parameters that can be used to measure the concentration or the rate of change of the concentration. To this end, the sensor 107 is arranged such that the sensor 107 can detect one or more respective parameters regarding the contents of the retort 103. For example, the sensor 107 can be arranged on a wall of the retort 103 or can project into the interior space of the retort 103.

[0028] For example, the sensor can be a device for detecting the speed of sound and / or the impedance of the liquid to be tested. These parameters vary with the concentration of different chemical species in the mother liquor and can be measured very precisely. However, other types of sensors can also be used as sensor 107, in particular sensors that allow concentration measurements (e.g. pH electrodes, spectrometers, etc.).

[0029] From the parameters thus measured, the tissue processor's computing unit 150 can determine the concentration or rate of change of concentration in the retort. The determination of the concentration or rate of change of concentration can in some embodiments be performed entirely in the sensor 107, for example using a dedicated computing unit of the sensor, which can be provided, for example, in the form of an integrated circuit, a microprocessor or other device suitable for data processing.

[0030] The concentration change rate can also be determined by determining (measuring) the absolute concentration and correlating it with an evaluation of its time course. For this purpose, for example, a derivative of the time course of the concentration (time course of the concentration) can be formed, which is suitably filtered and / or smoothed. It may also be possible to evaluate (determine) the concentration change rate without determining (measuring) the actual concentration, since for this purpose, in principle, the raw data course of the sensor signal generated by the sensor 107 can also be taken into account. If the sensor signal is constant, its rate of change is equal to zero, since the concentration on which it is based is also constant. In contrast, if the rate of change of the sensor signal is large, it can be assumed (judged) that the concentration change rate is also large.

[0031] Local concentration differences inside the retort 103 can be eliminated by proper thorough mixing, for example with a stirrer (not shown), to avoid fluctuations in the measurements of the sensor 107.

[0032] To facilitate (passive) reagent exchange at the tissue interface, the retort 103, and therefore the introduced liquid, can be heated. This enhances the Brownian molecular motion and thereby the corresponding diffusion rate that ultimately governs penetration into the tissue 105.

[0033] In FIG. 2, typical concentration changes such as may occur in a liquid within the framework of the present invention are shown in a simplified form in the form of a concentration-time graph and are labelled c(A) and c(B). In this case, the horizontal axis forms the time axis, while the vertical axis indicates the respective concentrations. It should be clearly noted that the position and scaling of both concentration curves c(A) and c(B) relative to one another is merely for illustrative purposes and does not allow drawing any conclusions (determining) about the respective absolute or relative concentrations of the species concerned. It should be noted in particular that the axis intersections do not necessarily coincide with the zero value of the respective axis. In contrast, the relative changes within both concentration profiles c(A) and c(B) are correctly shown: species A is the element (component) contained in the liquid concerned and introduced into the tissue to be treated, thus decreasing its concentration within the liquid during the course of the respective method step, until an equilibrium is established. Species B, on the other hand, is a compound that is present or formed inside the tissue 105 and that migrates from the tissue into the liquid during the respective method steps, so that its concentration in the liquid continues to increase during the method steps until the concentrations in the tissue 105 and the liquid are approximately equal or an equilibrium is established. An asymptotic change (progression) is obtained in both concentration changes c(A) and c(B) because the driving force of the concentration change, i.e. the concentration difference between the tissue 105 and its surrounding liquid, decreases over time. The smaller the concentration difference in question, the smaller the corresponding net diffusion rate that reduces the concentration difference, resulting in a decrease in the slope of the respective concentration curves c(A), c(B).

[0034] During operation of an advantageous embodiment of a method of the invention that can be implemented in the tissue processing device or tissue processing device 100, the tissue sample 105 is successively exposed to a number of different processing media 110, 120, 130, 140 in the form of liquids (fluids), in which, in the example shown, a fixative 110 is applied first, which serves to harden the tissue. For example, formaldehyde, paraformaldehyde, glutaraldehyde, other aldehyde, acetone or alcoholic fixatives and their solutions and / or mixtures can be used for this purpose. Usually, a 3.7% formalin solution (3.7% aqueous formaldehyde buffered to neutral pH with phosphate) is used for this purpose. In this case, the fixative 110 can be applied once or several times to the same sample 105. When the tissue is introduced into this fixative, for example the formalin solution mentioned above, the formaldehyde concentration of the solution initially decreases over time. This is because the tissue absorbs and consumes the fixative (cross-linking of proteins). Such a decrease in concentration c(A), as described above, is shown diagrammatically in Figure 2.

[0035] Fixation 110 is followed by one or more steps of liquid exchange with an exchange solvent 120. For this, the tissue sample 105 is immersed in the exchange solvent (or the fixative is removed from the retort and the exchange solvent is introduced into the retort), so that the exchange solvent 120 completely covers the sample 105. This expels any water still present from the sample 105, dissolves fat and washes away the residues of the fixative 110. The concentration of the main components of the exchange solvent 120 thus decreases in line with the concentration curve c(A) described above, while the concentration of species dissolved or expelled from the tissue 105 (fat, water, fixative 110) in the exchange solvent 120 increases according to the concentration curve c(B) shown in FIG. 2. As exchange solvents, inter alia, ethanol, methanol, isopropanol and other aliphatic and non-aliphatic alcohols and mixtures thereof come into consideration, for example 70% ethanol. What is important is the miscibility of the exchange solvent 120 used with water or the relatively high solubility of water in the exchange solvent 120 used. The liquid exchange can also be carried out in multiple steps, e.g. 2, 3, 4, 5 or even 10 or 20 steps, but in order to reduce costs and still achieve high quality processing, it is preferred to use a higher purity exchange solvent 120 in the later steps than in the earlier steps. Notably, the exchange solvent 120 in the final step of the liquid exchange can be anhydrous.

[0036] One practical example: the tissue 105, previously immersed in a formalin solution 110 (96.3 vol.% water), is placed in, for example, 70% ethanol 120 in a second step. Here, the water in the tissue 105 is exchanged with the surrounding ethanol 120. This causes a decrease in the ethanol concentration (c(A)) in the exchange medium 120 surrounding the tissue 105. This does not decrease further after some time t [s]. The tissue 105 then has substantially the same ethanol concentration as the exchange medium 120. The sensor 107 detects this substantially asymptotic decrease in concentration c(A) as explained above. When the concentration of the exchange medium 120 has stabilized, it is, for example, pumped out of the retort 103 or replaced.

[0037] Conversely, tissue 105 placed in, for example, ethanol 120 will also release substances into the reagent over time, for example fats and certain proteins. When the amount of these released components (concentration curve c(B) in FIG. 2) no longer increases further, it can be inferred that complete tissue infiltration has occurred after a certain time t [s]. The sensor then monitors this increase in amount, and the system can subsequently start the next process step. This can be detected particularly well in the first steps of the liquid exchange, since these substances are then quantitatively dissolved and a corresponding change in concentration of these substances can no longer be expected.

[0038] In the illustrated example, after the liquid exchange with the [exchange solvent] 120, the tissue 105 is clarified using a clarified liquid 130 which replaces the exchange solvent 120 inside the tissue 105. As clarified liquid, isopropanol, chloroform, xylene, toluene and other aromatic hydrocarbons as well as mixtures and non-aqueous solutions thereof can be used, among others. Here too, the miscibility or solubility (solubility) of the exchange solvent 120 used in the clarified liquid 130 is important. In the case of clarification, multiple steps are also possible, for example 2, 3, 4 or more steps, in which case the different clarification steps can be carried out with different clarified liquids 130 (in terms of identity, purity and / or mixture ratio). In particular, in the early clarification steps(s), a clarified liquid 130 with a relatively high polarity can be used, and in the later clarification steps, a clarified liquid with a lower polarity can be used. This allows a better miscibility with the previous or subsequent liquids, respectively.

[0039] Next, the tissue 105 is impregnated with the impregnating agent 140. In particular, the impregnating agent can be a mixture of various branched and / or unbranched alkanes with suitable additives. In this case, it is particularly preferred to use an impregnating agent that forms a solid with sufficient strength (stability) at temperatures below 20° C. and above −70° C., in particular above −20° C., to allow thin sectioning of the composite of the tissue 105 and the cooled impregnating agent 140.

[0040] In one embodiment of such a method according to the invention, in at least one of the above steps, the rate of change of concentration of the corresponding liquid 110, 120, 130, 140 is determined (measured). For this purpose, a sensor 107 is used. For example, during the liquid exchange, the change in concentration of water in the respective exchange medium 120 can be determined (measured). This rate of change of concentration naturally decreases in the course of each step until an equilibrium concentration is established, as already explained with reference to FIG. 2. After the equilibrium concentration is established, no further concentration changes can be expected any more, so that the rate of change of concentration (the slope of the respective concentration curves c(A), c(B) in FIG. 2) falls to zero. The rate of change of concentration can therefore be used to detect the virtual end of a process step (or steps). If the rate of change falls below a preset threshold, which may in particular be close to zero, the respective step can be considered to be ended. Then, preferably, a signal is output prompting the user of the tissue processing device to start the next step in each case, or the next step is started automatically, for example by replacing the liquid currently present in the retort 103 with another liquid used for the next step.

[0041] In practice, the concentration change rate can also be determined by comparing the currently determined measured value with the immediately preceding measured value (e.g. by taking the quotient or difference). Depending on the comparison result, it can be considered that a threshold value for the concentration change rate has been reached. For example, a quotient having a small distance from 1 (close to 1) or a corresponding difference having a small distance from 0 (close to 0) can be used as a corresponding indicator.

[0042] In this regard, all steps can be performed in the same retort 103, so that the retort can have an outlet and an inlet, for example, for removal of the previous liquid and for filling with the next liquid. In such configurations, no manipulation of the treated tissue 105 is necessary, so that the risk of damage to the tissue 105 is minimized. In alternative configurations, each of the steps can be performed in its own dedicated retort 103, so that, for example, a cage can be used to receive the tissue 105, but the cage can be brought to each retort 103 in each case to contact the tissue 105 with the respective liquid.

[0043] Monitoring of concentrations or rates of change thereof may generally relate to all compounds present in each fluid, such as gases dissolved or suspended in the fluid, major components of each fluid, and / or substances that have migrated into the fluid from tissue 105.

[0044] In any case, when the concentration change rate approaches zero, the end of the respective process step can be estimated (anticipated), so that no determination (measurement) of the absolute concentration is necessary. This, as already explained, has the advantage of a significantly simpler data processing, since in some cases it is possible not to perform a calibration of the sensor 107 for the respective liquid and the compound to be analyzed, and thus no conversion (conversion) of the sensor signal to the respective concentration is necessary. For the evaluation of the concentration change rate, it is sufficient to take into account the raw signal in each case. In any case, different thresholds are required for the different process steps in order to take into account the absolute concentration changes that are to be anticipated in each case. For example, a significantly higher concentration change rate can be expected in the first step of a liquid exchange than in the last step, since towards the end of the liquid exchange, there is already an almost pure exchange medium 120 present inside the sample, and therefore the concentration difference between the tissue 105 and the exchange medium 120 is relatively small.

[0045] This monitoring of the concentration change rate can be applied to each of the previously described method steps, thus allowing a large degree of automation of the tissue processing as a whole. Conventionally, there is a problem that different sample sizes require very different processing times, which must be determined empirically. Within the scope of the present invention, by monitoring the concentration change rate, the optimal processing time in each case is automatically obtained, thus ensuring the same processing quality.

[0046] Typically, in order to be able to derive reliable predictions from the concentration change rate regarding the progress of the process, the ratio of tissue volume to the volume of the liquid used should not leave the range of 1:1 to 1:50. If the tissue volume is too small compared to the liquid volume, the concentration change rate may not be detected with sufficient accuracy.

[0047] As mentioned in the introduction, this type of monitoring of the rate of concentration change can also be used to control other treatments of the biological tissue 105. For example, the staining of a particular tissue portion can be monitored and controlled by monitoring the concentration of the staining substance in the staining solution, since in this case the rate of concentration change continues to decrease.

[0048] Furthermore, independently of the specific form of the respective process step, the depletion of a solution or liquid of a substance used in a process step can be ascertained by the fact that the rate of change at the start of the respective process step is smaller than a predefinable threshold. In particular, the threshold representing an excessively small concentration of a liquid can depend on the sample size of the tissue 105 to be processed, since a small rate of change in concentration can be expected when the sample size is small. The same can be true for the threshold used to determine the end of a process step. Thus, a rate of change in concentration below a predefinable threshold at the start of a process step can, for example, cause or trigger the replacement of the liquid concerned.

[0049] It should be emphasized here that the features and their respective advantages can be realised not only in the combinations shown, but also in other combinations and possibly alone, without departing from the scope of the invention.

[0050]

Claims

1. A method for tissue processing of at least one biological tissue (105), comprising the steps of: Treatment of at least one tissue with at least one liquid (110, 120, 130, 140); Measuring the rate of change of concentration in the at least one liquid (110, 120, 130, 140); and Taking action depending on the measured rate of change of concentration A method comprising:

2. 10. The method of claim 1 , said treatment including at least partially staining said at least one tissue (105); A method comprising:

3. The method according to claim 1 or 2, The processing of the at least one tissue (105) comprises, in sequence, the following steps: Fixing the at least one tissue (105) with at least one fixative (110), whereby the at least one tissue (105) is hardened. Fluid exchange with a polar non-aqueous exchange solvent (120), wherein water is displaced from said at least one tissue (105) and replaced by said exchange solvent (120); clarification with a clarification solution (130), wherein the exchange solvent (120) in the at least one tissue (105) is replaced by the clarification solution (130); and impregnation of said at least one system (105) with an impregnation agent (140), wherein said clear liquid (130) in said at least one system (105) is replaced by said impregnation agent (140); Inclusion of one or more of the following in a purposive order: - measurements of the rate of change of concentration in the fixative solution (110) and / or the exchange solvent (120) and / or the clear solution (130) and / or the impregnating agent (140) are carried out during their respective uses; and performing at least one action dependent on at least one of the measured rates of concentration change; A method comprising:

4. In the method according to claim 3, The clarified liquid (130) comprises an organic solvent that is less polar than the exchange solvent (120). A method comprising:

5. In the method according to any one of claims 1 to 4, said measures include terminating each method step during which the concentration change rate was measured and proceeding to the respective next method step if the concentration change rate falls below a predefinable threshold value; A method comprising:

6. The method according to any one of claims 1 to 5, The method further comprises: Measurement of at least one absolute concentration in the liquid; and Execution of replacement of the respective fluids (110, 120, 130, 140) when the measured absolute concentration reaches a predefinable threshold value. Contains A method comprising:

7. The method according to claim 3 or 4, The method further comprises: - measuring at least one absolute concentration in the fixative solution (110) and / or in the exchange solvent (120) and / or in the clarified solution (130) and / or in the impregnating agent (140); and Execution of replacement of the respective fluids (110, 120, 130, 140) when the measured absolute concentration reaches a predefinable threshold value. Contains A method comprising:

8. 1. A tissue processing apparatus comprising: The tissue processing device (100) comprises: providing at least one sensor (107) configured to measure concentrations and / or rates of change of concentrations in liquids (110, 120, 130, 140) used in the tissue processing device (100); The tissue processing device (100) is adapted to carry out the method according to any one of claims 1 to 7. A tissue processing device comprising:

9. 9. The tissue processing device of claim 8, the sensor (107) is configured to measure concentration and / or rate of change of concentration based on the speed of sound in the liquid (110, 120, 130, 140) and / or the acoustic impedance of the liquid (110, 120, 130, 140) and / or the temperature of the liquid (110, 120, 130, 140) and / or the chemical and / or electrochemical and / or electrical behavior of the liquid (110, 120, 130, 140) and / or the optical properties of the liquid (110, 120, 130, 140). A tissue processing device comprising:

Images