TISSUE PROCESSING METHOD AND TISSUE PROCESSOR

Description

[0001] The present invention relates to a method for tissue processing of at least one biological tissue and to a tissue processor usable for this purpose. Background of the invention

[0002] Tissue processors can be used to prepare biological tissues for pathological examinations, for example. Such tissue processors are described, for instance, in DE 10 2009 038 481 A1 and DE 10 2008 054 071 A1.

[0003] WO 2019 / 036760 A1 describes a tissue processor in which the purity of a processing fluid is determined by sensory means and is used or discarded for tissue processing based on the determined purity. Disclosure of the invention

[0004] According to the invention, a method for tissue processing and a tissue processor with the features of the independent claims are proposed. Advantageous embodiments are the subject of the dependent claims and the following description.

[0005] In detail, a tissue processing method according to the invention comprises processing the at least one tissue using at least one fluid, determining a concentration change rate in the at least one fluid, and performing an action depending on the determined concentration change rate. This allows a meaningful end to be determined for each processing step (as the concentration change rate decreases, the tissue undergoes less chemical change) and an appropriate action to be initiated. For example, the action could be a signal output to indicate the end of the processing step, or a subsequent processing step could be started automatically.

[0006] In particular, the processing can include at least partial staining of at least one tissue. This prevents excessive staining, which has a positive effect on the visibility of tissue features under examination.

[0007] Alternatively or additionally, the processing of the at least one fabric may successively comprise one or more of the following steps, expediently in the order given: fixing the at least one fabric using at least one fixing fluid, wherein the at least one fabric is hardened; fluid exchange using a polar non-aqueous exchange solvent, wherein water is displaced from the at least one fabric and replaced by the exchange solvent; clarifying using a clarifying fluid, which in particular comprises a less polar organic solvent than the exchange solvent, wherein the exchange solvent in the at least one fabric is replaced by the clarifying fluid; and impregnating the at least one fabric using an impregnating agent, wherein the clarifying fluid in the at least one fabric is replaced by the impregnating agent.The rate of concentration change in the fixative fluid and / or the exchange solvent and / or the clarifying fluid and / or the impregnating agent is determined during each use, and at least one action is taken depending on at least one of the determined rates of concentration change. This ensures consistent preparation quality for tissue samples, regardless of their size, geometry, or other properties. Excessive or insufficient infiltration of the respective fluid into the tissue is thus reliably avoided.

[0008] The measure can, in particular, include terminating the respective process step during which the concentration change rate was determined and proceeding to the next process step if the concentration change rate falls below a predetermined threshold. This enables a high degree of automation in tissue preparation.

[0009] For example, the threshold for the concentration change rate can be less than 20, 10, 5, or 2% / min, based on an absolute concentration of the respective species. At such low change rates, no significant impact on the overall quality of tissue processing is expected. Alternatively or additionally, the threshold can be determined based on the size, mass, or volume of at least one tissue sample to ensure consistent processing conditions even with varying sample sizes. Incorporating quality requirements can also be advantageous, allowing for a lower threshold for high requirements and a higher threshold (and consequently a shorter processing time) for lower quality requirements.It should be noted that the concentration changes under consideration typically follow an exponential function, so that a doubling of the threshold will typically not be accompanied by a halving of the processing time.

[0010] In advantageous embodiments, the process further comprises determining at least one absolute concentration in the fluid, in particular the fixer and / or the exchange solvent and / or the clarifying fluid and / or the impregnating agent, and initiating an exchange of the respective fluid when the determined absolute concentration reaches a predefinable threshold value. This ensures a minimum quality of the fluid(s) and thus optimizes the process economically with regard to fluid utilization without compromising quality.

[0011] It should be emphasized here that each of the processing steps mentioned can also comprise several, essentially identical, individual steps. For example, fluid exchange can be achieved through several, for example, 2, 3, 4, 5 or more successive baths in the respective exchange solvent. The same applies to the other processing steps mentioned.

[0012] A typical fixative contains 1 to 4% formaldehyde in aqueous solution and is stabilized against autopolymerization with 0.5 to 2% methanol. Furthermore, the fixative may contain a phosphate buffer (e.g., potassium dihydrogen phosphate and / or disodium hydrogen phosphate dihydrate, 1 to 12 g / 1000 mL).

[0013] The exchange solvent typically contains a mixture of a simple alcohol (e.g., ethanol and / or isopropanol) and water with an alcohol content of 10 to 99.9%. During fluid or media exchange, lipids, especially fats and sphingolipids (particularly ceramides), and lipoproteins are typically dissolved from the tissue. Especially towards the end of the first fluid exchange step, these substances can constitute a total of 1 to 15% of the exchange solvent.

[0014] The clarifying fluid typically contains xylene and / or other aromatic hydrocarbons (e.g., toluene) as well as a simple alcohol (e.g., the alcohol(s) also contained in the exchange solvent), with the proportion of aromatic species in the clarifying fluid ranging from 10 to 99.9%.

[0015] Histological paraffin is generally used as the impregnating agent, which may contain the aromatic substances present in the clarifying fluid. The proportion of paraffin in the impregnating agent ranges from 70 to 99.9%. All concentration and proportion values ​​refer to the volume of the mixture under consideration. Typically, concentrations can increase from several baths of a single processing step to subsequent baths.

[0016] A tissue processor according to the invention comprises at least one sensor configured to determine a concentration and / or a rate of concentration change in a fluid used in the tissue processor, and means enabling the tissue processor to carry out a method as described above. The tissue processor thus benefits analogously from the advantages of a corresponding method as explained above, and vice versa. The sensor according to the invention is defined in claim 6.

[0017] The sensor is preferably configured to determine the concentration and / or rate of concentration change based on the speed of sound in the fluid and / or the acoustic impedance of the fluid and / or the temperature of the fluid and / or the chemical and / or electrochemical and / or electrical behavior of the fluid and / or the optical properties of the fluid. These are particularly suitable parameters from which precise conclusions can be drawn about fluid concentrations and / or their rates of change.

[0018] Further advantages and embodiments of the invention will become apparent from the description and the accompanying drawing.

[0019] It is understood that the features mentioned above and those to be explained below can be used not only in the combinations specified, but also in other combinations or on their own, without leaving the scope of the present invention.

[0020] The invention is schematically illustrated in the drawing using an exemplary embodiment and is described below with reference to the drawing. Reference numerals that refer to a device component are also used to designate a process step carried out therein, and vice versa, to avoid repetition. Character description

[0021] Figure 1 shows a schematic representation of an advantageous embodiment of a tissue processor according to the invention. Figure 2 shows typical concentration profiles such as can occur in fluids within the scope of the present invention.

[0022] In Figure 1 An advantageous embodiment of a tissue processor according to the invention is shown schematically and is generally designated by 100.

[0023] The tissue processor 100 comprises at least one retort 103, which is suitable and equipped for receiving one or more biological tissues or samples 105 and for filling with at least one organic fluid. In particular, the retort may comprise one or more materials from the group consisting of stainless steel, aluminum and other metals, polyethylene etherketone (PEEK), polytetrafluoroethylene (PTFE), polyethylene (PE), polypropylene (PP), polycarbonate (PC) and other suitable plastics and glass.

[0024] Furthermore, the tissue processor 100 includes a sensor 107, which is configured to determine a concentration and / or a concentration change rate and / or other parameters that can be used to determine a concentration or concentration change rate. For this purpose, the sensor 107 is arranged so that it can detect the respective parameter(s) with respect to a content of the retort 103. For example, the sensor 107 can be arranged in a wall of the retort 103 or project into an inner volume of the retort 103.

[0025] For example, the sensor could be a device that detects the speed of sound and / or the impedance of the fluid under investigation. These parameters change with the concentration of different chemical species in a matrix liquid and can be measured very precisely. However, other sensor types besides sensor 107 can also be used, especially those that enable concentration measurement (e.g., pH electrodes, spectrometers, or the like).

[0026] From such measured parameters, a processing unit 150 of the tissue processor can determine the concentration or rate of concentration change in the retort. In some embodiments, the determination of the concentration or its rate of change can also be performed integrally within the sensor 107, for example, using a dedicated processing unit of the sensor, which may be provided, for example, in the form of an integrated circuit, a microprocessor, or other suitable data processing devices.

[0027] A concentration change rate can also be determined by ascertaining an absolute concentration in conjunction with an analysis of its temporal profile. For example, this can be achieved by deriving a suitably filtered and / or smoothed temporal concentration profile. Assessing a concentration change rate without determining an actual concentration is also possible, as the raw data profile of a sensor signal generated by sensor 107 can, in principle, be used. If the sensor signal is constant, the underlying concentration is also constant, and therefore its change rate is zero. Conversely, high change rates in the sensor signal can be assumed to indicate a high concentration change rate.

[0028] Local concentration differences within the retort 103 can be reduced by suitable mixing, for example by means of a stirrer (not shown in the figure), in order to avoid fluctuations in the measurements of the sensor 107.

[0029] To accelerate the (passive) exchange of reagents at the tissue interface, the retort 103 and thus the fluids used can be heated. This intensifies Brownian motion and therefore increases the corresponding diffusion rates, which are ultimately responsible for the infiltration of tissue 105.

[0030] In Figure 2Typical concentration profiles, such as those that can occur in fluids within the scope of the present invention, are simplified and represented in the form of a concentration-time diagram, labeled c(A) and c(B), respectively. The abscissa represents the time axis, while the ordinate indicates the respective concentration. It should be expressly noted that the positioning and scaling of the two concentration profiles c(A) and c(B) relative to each other serves a purely illustrative purpose and does not allow any conclusions to be drawn about the respective absolute or relative concentrations of the species in question. In particular, it should be noted that the intersection point of the axes does not necessarily coincide with a zero value on the respective axis.The internal relative profile of both concentration profiles c(A) and c(B) is correctly represented: Species A is a component contained in the fluid and introduced into the processed tissue 105, causing its concentration within the fluid to decrease during each process step until equilibrium is reached. Species B, on the other hand, is a compound present or formed within tissue 105 and migrates from the tissue into the fluid during each process step, increasing its concentration in the fluid until the concentrations in tissue 105 and the fluid are equalized or equilibrium is reached.Both concentration profiles c(A) and c(B) exhibit an asymptotic course, as the driving force of the concentration change, namely the concentration difference between tissue 105 and the surrounding fluid, decreases over time. The lower the concentration difference, the lower the corresponding net diffusion rate that reduces the concentration difference, resulting in a decreasing slope for the respective concentration profiles c(A) and c(B).

[0031] In the operation of the tissue processor, or in an advantageous embodiment of a method according to the invention, which can be implemented in the tissue processor 100, the tissue sample 105 is successively exposed to several different process media 110, 120, 130, 140 in the form of fluids. In the example shown here, a fixative fluid 110 is applied first, which serves to harden the tissue. For example, formaldehyde, paraformaldehyde, glutaraldehyde, other aldehydes, acetone, or alcoholic fixatives, as well as solutions and / or mixtures thereof, can be used for this purpose. A 3.7% formalin solution (3.7% formaldehyde solution in water buffered to a neutral pH with phosphate salts) is typically used for this purpose. The fixative fluid 110 can be applied once or several times to the same sample 105.When tissue is immersed in this fixative fluid, for example the aforementioned formalin solution, the formaldehyde concentration of the solution initially decreases over time because the tissue absorbs and consumes it (cross-linking of proteins). Such a decrease in concentration c(A) is, as described above, in . Fig. 2 schematically represented.

[0032] Following fixation 110, one or more fluid exchange steps with an exchange solvent 120 are performed. For this purpose, the tissue sample 105 is immersed in the exchange solvent (or the fixative fluid is removed from the retort and exchange solvent is introduced into the retort) so that the exchange solvent 120 completely covers the sample 105. This displaces any remaining water from the sample 105, dissolves fats, and washes away residues of the fixative fluid 110. Therefore, the concentration of the main component of the exchange solvent 120 decreases analogously to the concentration profile c(A) described above, while the concentration of species dissolved or displaced from the tissue 105 (fats, water, fixative fluid 110) in the exchange solvent 120 decreases according to the profile described above. Fig. 2The concentration profile c(B) shown increases. Suitable exchange solvents include, in particular, ethanol, methanol, isopropanol, and other aliphatic and non-aliphatic alcohols, as well as mixtures thereof, e.g., 70% ethanol. Crucially, the exchange solvent 120 must be miscible with water or have a relatively high solubility of water in the exchange solvent 120. The fluid exchange can also be carried out in several steps, for example, 2, 3, 4, 5, or up to 10 or 20. Preferably, a higher purity exchange solvent 120 is used in later steps than in earlier steps to reduce costs while still achieving high processing quality. In particular, the exchange solvent 120 used in the final step of the fluid exchange can be anhydrous.

[0033] Practical example: Tissue 105, which was previously infiltrated in formalin solution 110 (96.3 vol% water), is introduced in a second step into, for example, 70% ethanol 120. Here, the water of the tissue 105 is exchanged for the surrounding ethanol 120. This reduces the ethanol concentration (c(A)) in the exchange solvent 120 around the tissue 105. After a certain time t[s], this concentration stops decreasing. The tissue 105 then has an essentially identical ethanol concentration to the exchange solvent 120. As already explained, the sensor 107 detects this essentially asymptotic decrease in concentration c(A). Once the concentration of the exchange solvent 120 has stabilized, it is pumped out of the retort 103 or replaced.

[0034] Tissue 105, introduced into ethanol 120, also releases substances into the reagent over time, e.g., fat and certain proteins. As the amount of substance (concentration profile c(B)) decreases in Fig. 2 Since the amount of these released components does not increase further, complete tissue infiltration can be assumed after a time t[s]. The sensors monitor this increase in quantity, and the system can then start the next process step. This is particularly easy to detect in the initial stages of fluid exchange, as these substances are then quantitatively dissolved and no further change in their concentration is expected.

[0035] Following fluid exchange with 120, the tissue 105 is clarified in the illustrated example using a clarifying fluid 130, which replaces the exchange solvent 120 within the tissue 105. The clarifying fluid 130 can be, in particular, isopropanol, chloroform, xylene, toluene, and other aromatic hydrocarbon compounds, as well as mixtures thereof and non-aqueous solutions. Here, the miscibility or solubility of the exchange solvent 120 in the clarifying fluid 130 is again crucial. The clarifying process can also be carried out in several steps, for example, two, three, four, or even more, with different clarifying fluids 130 being used for each step (with respect to identity, purity, and / or mixing ratio).In particular, a clarification fluid 130 with relatively high polarity can be used in the early stages of the clarification process, while a clarification fluid with lower polarity can be used in later stages. This results in better miscibility with the fluid used before or after, respectively.

[0036] Subsequently, the fabric 105 is impregnated with an impregnating agent 140. In particular, mixtures of various branched and / or unbranched alkanes with suitable additives can be used as the impregnating agent. Impregnating agents that form solids with sufficient strength at temperatures below 20 °C and above -70 °C, and especially also at temperatures above -20 °C, to allow thin sections of a composite of fabric 105 and cooled impregnating agent 140 are particularly preferred.

[0037] In an embodiment of such a method according to the invention, a concentration change rate in the corresponding fluid 110, 120, 130, 140 is determined in at least one of the described steps. For this purpose, the sensor 107 is used. For example, a concentration change of water in the respective exchange solvent 120 can be determined during the fluid exchange. This concentration change rate, as already mentioned with reference to Fig. 2 As explained, the concentration naturally decreases during each step until an equilibrium concentration is reached. Once the equilibrium concentration is reached, no further change in concentration is to be expected; the rate of concentration change (slope of the respective concentration curves c(A), c(B) in Fig. 2The concentration change rate thus drops to zero. Therefore, the rate of concentration change can be used to detect the actual end of a process step. If the rate falls below a predefined threshold, which can be close to zero, the respective step can be considered complete. Preferably, a signal is then output that prompts a user of the tissue processor to initiate the next step, or the next step is initiated automatically, for example, by replacing the fluid currently in retort 103 with a different fluid used for the subsequent step.

[0038] In practice, the rate of concentration change can also be determined by comparing a currently measured value with the immediately preceding measured value (e.g., using a quotient or a difference). Depending on the comparison result, the threshold for the rate of concentration change can be considered to have been reached. For example, a quotient that is close to 1 or a corresponding difference that is close to 0 can be used as a suitable indicator.

[0039] It should be mentioned in this context that all steps can be carried out in one and the same retort 103, so that, for example, an outlet opening and an inlet opening can be provided in the retort for removing the previous fluid and filling in the subsequent fluid. In such embodiments, no manipulation of the processed fabric 105 is required, thereby minimizing the risk of damaging the fabric 105. In alternative embodiments, each of the steps can be carried out in a separate retort 103, in which, for example, a basket can be used to hold the fabric 105, so that the basket can be inserted into the respective retort 103 to establish contact between the fabric 105 and the respective fluid.

[0040] Monitoring the concentration or its rate of change can generally concern all compounds present in the respective fluid, for example, gases dissolved or suspended in a fluid, a main component of the respective fluid and / or substances passing from tissue 105 into the fluid.

[0041] In any case, the end of the respective process step can be assumed when the concentration change rate approaches zero, so that determining an absolute concentration is unnecessary. As already explained, this offers the advantage of significantly simpler data processing, since calibration of sensor 107 for the specific fluid and compound being analyzed can be omitted, and conversion of the sensor signals into the respective concentrations is unnecessary. Examining the raw signal is sufficient to assess the concentration change rate. At most, different threshold values ​​for different process steps may be required to account for the expected absolute concentration changes.For example, in the first step of the fluid exchange, significantly higher concentration change rates can be expected than in the last step, since towards the end of the fluid exchange, almost pure exchange solvent 120 is already present within the sample and the concentration difference between tissue 105 and exchange solvent 120 is therefore comparatively small.

[0042] This monitoring of concentration change rates can be applied to each of the previously described process steps, thus enabling a high degree of automation in tissue processing. Traditionally, the problem arises that different sample sizes require very different processing times, which must be determined empirically. Within the scope of the invention, the optimal processing time is automatically determined by monitoring the concentration change rates, thereby ensuring consistent processing quality.

[0043] Typically, the ratio of tissue volume to fluid volume should not exceed 1:1 to 1:50 in order to derive reliable conclusions about process progress from the concentration change rate. If the tissue volume is too small compared to the fluid volume, the concentration change rate cannot be measured with sufficient accuracy.

[0044] As mentioned at the outset, such monitoring of concentration change rates can also be used to control other processing of biological tissue. For example, the staining of specific tissue parts can be monitored and controlled by monitoring the dye concentration in a staining solution, since the concentration change rate also decreases steadily in this case.

[0045] Furthermore, regardless of the specific design of the respective process step, a depletion of solutions or fluids in terms of substances consumed in a process step can be detected by the fact that the rate of change at the beginning of the respective process step is lower than a predetermined threshold value. In particular, such a threshold value, which describes an insufficient concentration of a fluid, can depend on the sample size of the processed tissue, since a small sample size is expected to result in a lower rate of concentration change. The same principle can apply to the threshold value used to determine the end of a process step. Therefore, if the rate of concentration change falls below a predetermined threshold value at the beginning of a process step, a replacement of the fluid in question can be triggered or initiated.

[0046] It should be emphasized here that the features and their respective advantages can be realized not only in the specified combination, but also in other combinations and, if necessary, on their own, without leaving the scope of the present invention.

Claims

1. Method for tissue processing of at least one biological tissue (105), comprising processing the at least one tissue in a retort (103) using at least one fluid (110, 120, 130, 140), determining a rate of change in concentration in the at least one fluid (110, 120, 130, 140), and implementing a measure depending on the determined rate of change in concentration.

2. Method according to claim 1, wherein processing comprises at least partially dyeing the at least one tissue (105).

3. Method for tissue processing of at least one biological tissue according to claim 1 or 2, wherein processing the at least one tissue (105) sequentially comprises one or a plurality of the following steps, expediently in the specified order: fixing the at least one tissue (105) using at least one fixing fluid (110), wherein the at least one tissue (105) is cured, exchanging a fluid using a polar non-aqueous exchange solvent (120), wherein water is displaced from the at least one tissue (105) and replaced by the exchange solvent (120), clarifying using a clarifying fluid (130), which in particular comprises a more non-polar organic solvent than the exchange solvent (120), wherein the exchange solvent (120) in the at least one tissue (105) is replaced by the clarifying fluid (130), and impregnating the at least one tissue (105) using an impregnating agent (140), wherein the clarifying fluid (130) in the at least one tissue (105) is replaced by the impregnating agent (140), wherein determining the rate of change in concentration in the fixing fluid (110) and / or the exchange solvent (120) and / or the clarifying fluid (130) and / or the impregnating agent (140) is carried out during the respective use, and at least one measure is carried out depending on at least one of the determined rates of change in concentration.

4. Method according to any one of the preceding claims, wherein the measure comprises terminating the respective method step during which the rate of change in concentration was determined and proceeding to the respectively following method step if the rate of change in concentration falls below a predeterminable threshold value.

5. Method according to any one of the preceding claims, further comprising: determining at least one absolute concentration in the fluid, in particular with reference to claim 3 the fixing fluid (110) and / or the exchange solvent (120) and / or the clarifying fluid (130) and / or the impregnating agent (140), and initiating an exchange of the respective fluid (110, 120, 130, 140) when the determined absolute concentration reaches a predefinable threshold value.

6. Tissue processor (100) for treating tissue samples, comprising a retort, configured to receive one or a plurality of biological tissues or samples (105) and to fill them with at least one organic fluid, and at least one sensor (107), wherein the sensor is configured to determine a rate of change in concentration in a fluid (110, 120, 130, 140) used in the retort, and the tissue processor is configured to carry out further measures depending on the determined rate of change in concentration.

7. Tissue processor (100) according to claim 6, wherein the sensor (107) is configured to determine the concentration and / or rate of change in concentration based on a speed of sound in the fluid (110, 120, 130, 140) and / or an acoustic impedance of the fluid (110, 120, 130, 140) and / or a temperature of the fluid (110, 120, 130, 140) and / or a chemical and / or electrochemical and / or electrical behaviour of the fluid (110, 120, 130, 140) and / or optical properties of the fluid (110, 120, 130, 140).