Patent Application
20240344943
This disclosure is directed to the field of tissue processing and methods thereof, for example, that can be used in high throughput applications for histology.
Conventional histology processing protocols consist of four groups of reagents to provide fixation, dehydration, clearing, and infiltration. Of these, it is common to use catalysts such as heat, pressure, and/or vacuum on these protocols that usually consist of 12 steps. Some processors are equipped with a stirrer or some type of option to agitate the fluid. The shortcomings of traditional processors make it extremely difficult to eliminate heat or increase the rate of fluid perfusion. Therefore, there remains a need for improved methods of tissue processing that address and overcome these shortcomings while providing comparable or even superior results and still remaining compatible with standard reagents, equipment, and staining protocols.
Provided for herein is a method of processing a tissue sample, for example an accelerated protocol, for use in histology.
For example, a method of processing a tissue sample of this disclosure comprises the steps of (i) to (xii):
It is to be noted that the term “a” or “an” entity refers to one or more of that entity; for example, “a tissue sample,” is understood to represent one or more tissue samples. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.
Furthermore, “and/or” where used herein is to be taken as specific disclosure of each of the specified features or components with or without the other. Thus, the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
It is understood that wherever aspects are described herein with the language “comprising” or “comprises” otherwise analogous aspects described in terms of “consisting of,” “consists of,” “consisting essentially of,” and/or “consists essentially of,” and the like are also provided.
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related.
Numeric ranges are inclusive of the numbers defining the range. Even when not explicitly identified by “and any range in between,” or the like, where a list of values is recited, e.g., 1, 2, 3, or 4, unless otherwise stated, the disclosure specifically includes any range in between the values, e.g., 1 to 3, 1 to 4, 2 to 4, etc.
The headings provided herein are solely for ease of reference and are not limitations of the various aspects or aspects of the disclosure, which can be had by reference to the specification as a whole.
As used herein, “ActivFlo” refers to a line of histology cassettes sold by Leica Biosystems (LBS) with a lateral vent design.
As used herein, “SPECTRA S2” is a validated Leica H&E Staining System.
As used herein, “HistoCore SPECTRA” is a Leica automated H&E Stainer.
As used herein, “HistoCore PELORIS 3” is a Dual Retort Rapid Tissue Processor.
As used herein, “neutral-buffered formalin (NBF)” refers to standard formulations comprising formaldehyde, water, and phosphate buffers known in the art, such as are commercially available. Some embodiments may also contain methanol. For example, some embodiments utilize 10% NBF, which is a standard in histology where a 10% solution of formaldehyde concentration (37% w/v) and water is prepared and buffered to maintain a pH of about 7.2.
Where the term “about” is used, e.g., about 30 minutes or about 70% ethanol, it is understood that the value recited is also included. Further, where the term “about” precedes multiple numbers, for efficiency, all the numbers are qualified by “about,” even if each number is not directly preceded by “about,” unless explicitly defined otherwise. For example, “a time of about 5, 10, 15, 20, 25, or 30 seconds” should be construed as “a time of about 5, about 10, about 15, about 20, about 25, or about 30 seconds.” For example, “a concentration between about a 6% and 14%” should be construed as “a concentration between about 6% and about 14%.”
As used herein, “drip” refers to the actual drip time following draining a reagent from a sample (and in some cases, from cassettes, biopsy pads, etc.).
Provided for herein is an accelerated tissue processing protocol, e.g., xylene tissue processing. While the accelerated tissue processing protocol of this disclosure is in a format common in histology, and compatible with commercially available tissue processors, what makes the system unique is that through a novel series of alcohol grading and time, the process is more efficient (i.e., takes less time) and requires less heat. In certain embodiments, the addition of agitating the sample (such as stirring) is used to enhance the efficiency and reduction of heat. Thus, the tissue processing protocol of this disclosure provides significant advantages in processing time.
For example, in certain embodiments, the accelerated protocol has a total processing time of less than about 6 hours, less than about 5.5 hours, or less than about 5 hours, or for example, less than about 6.5 hours, less than about 6 hours, or less than about 5.5 hours when fill and drain times are included. In certain embodiments, the accelerated protocol has a total processing time of about 5 hours or about 5.5 hours when fill and drain times are included. In contrast, comparable standard protocols for processing tissue samples currently require around 10 to 14 or more hours. Thus, in certain embodiments, the accelerated protocol has a total processing time that is at least about 33% less, 40% less, 50% less, 55% less, 60% less, or 67% less than a comparable standard protocol either compared with or without fill and drain times. Examples of comparable standard protocols include protocols for processing the same size and/or type of tissue sample and/or for the same downstream processing/histology use.
While not limiting, in certain embodiments, the protocol can be used as a factory protocol for the HistoCore PELORIS 3 Tissue Processor (LEICA BIOSYSTEMS MELBOURNE PTY LTD) (
While many different types of tissue cassettes can be used, it is noted that IP ActivFlo Routine I cassettes or similarly designed cassettes comprise particular lateral vents that in combination with a stirrer promote perfusion and temperature control. In certain embodiments, the cassette used is one described and/or claimed in U.S. Pat. No. 5,928,934, which is incorporated herein by reference.
In certain embodiments, the accelerated protocol utilizes paraffin and stirring, such as can be achieved using a HistoCore PELORIS 3 Tissue Processor. In certain embodiments, the protocol can be used with a processor with equivalent functionality to the HistoCore PELORIS 3 Tissue Processor, such as may be developed as a competitor to the HistoCore PELORIS 3 Tissue Processor or as LEICA BIOSYSTEMS or its successors may introduce at a later time (art recognized equivalent). In certain embodiments, the protocol can utilize cassettes similar to IP ActivFlo Routine I cassettes such as other versions of ActivFlo cassettes or such as may be developed as a competitor to IP ActivFlo Routine I cassettes or as LEICA BIOSYSTEMS or its successors may introduce at a later time (art recognized equivalent). In certain embodiments, such art recognized equivalent designs comprise lateral vents similar to the lateral vents of the IP ActivFlo Routine I cassette design.
The accelerated protocol is suitable for clinical and research applications. The disclosed methods provide for processing a tissue sample for use in histology. Applications include for clinical human and non-human, including companion animals, diagnostic and prognostic histology testing. Applications also include research applications for human and non-human samples. Such protocol can be used in processing a number of tissues, for example including but not limited to breast, colon, uterine, kidney, pancreas, liver, lung, skin, fat, muscle, connective tissue, bone, prostate, testicle, ovary, adrenal, thyroid, parotid, salivary gland, lymph node, and gastrointestinal samples.
The sample may be of a variety of dimension known to those skilled in the art of histology and related processing, such as for hematoxylin and eosin (H&E) staining and the like. For example for standard surgical specimens, sections can be prosected at a recommended maximum of 33×26×5 mm. Samples can be smaller, e.g., as small as 20×10×3 mm. Thus, in certain embodiments, the sample size can be anywhere in between about 20×10×3 mm and about 33×26×5 mm. For example, in certain embodiments, the sample has dimensions of about 25×20×3 mm. For example, in certain embodiments, the sample has dimensions of about 20×20×4 mm. One advantage of the present disclosure is that samples that exceed the recommended size can still be processed, often adequately, minimizing the risk of reprocessing (within the range of time).
The process can significantly eliminate the heat usually applied during the reagent steps, which better preserves nucleotides and sensitive epitopes. In certain embodiments, the process eliminates at least about 25%, 33%, 40%, 50%, 60%, or 70% of the heat usually applied during any individual step. In certain embodiments, the process eliminates up to about 75%, 80%, 85%, 90%, 95%, or up to around 100% of the heat usually applied during any individual step. In certain embodiments, the process eliminates at least about 25%, 33%, 40%, 50%, 60%, or 70% of the heat usually applied cumulatively during the process. In certain embodiments, the process eliminates up to about 67%, 75%, 80%, or 90% of the heat usually applied cumulatively during the process.
Certain embodiments provide for a twelve-step accelerated tissue processing protocol. One of ordinary skill in the art would recognize that common commercial tissue processing systems commonly utilize twelve-step protocols and thus the protocol of this disclosure is compatible with aspects of commonly used systems. One of ordinary skill in the art would recognize that fewer steps could be utilized but doing so may be counterproductive to efficiency as it could increase the time spent changing out expired reagents. However, using fewer steps in combination with the inventive features of this disclosure is also contemplated.
One aspect of the present disclosure is agitation of the reagents in contact with the tissue sample. Agitation can be applied by a variety of methods such as stirring, rocking, and swirling. One of ordinary skill in the art will recognize that the amount of agitation can be varied in various steps, for example, the stirring speed can be varied, e.g., a medium stirring speed in some steps and a high stirring speed in other steps. Altering the agitation between a high level and a lesser level can allow for the processing of delicate tissue that may not be able to withstand a high amount of agitation/stirrer speed during all steps. However, in some embodiments, a high level of agitation can be used for half or more (e.g., 6, 7, 8, 9, 10, 11, or 12) of the steps. In some embodiments, a high level of agitation can be used for all (e.g., 12) or nearly all (e.g., 9, 10, or 11) of the steps. Thus, certain embodiments require a setup that allows for varying the amount of agitation used. For example, the protocol is well-suited for use with the HistoCore PELORIS 3 Tissue Processor, because it comprises a large, heavy metal stirrer that has three speeds (factory protocols are defaulted to medium). In contrast, other commercial processors have only a small plastic stirrer at a set speed or no stirrer at all. However, it is understood that the protocol is not limited to use on a HistoCore PELORIS 3 Tissue Processor and could be used with any apparatus that provides for agitation/stirring or done manually. For example, on a tissue processor that provides for alteration between low, medium, and high stirring speeds.
Certain embodiments employ medium agitation. For example, in certain embodiments, the medium agitation is a stirring speed of about 59-60 rpm on a HistoCore PELORIS 3 Tissue Processor. In certain embodiments, the medium agitation is a stirring speed of about 59-60 rpm on equipment equivalent to a HistoCore PELORIS 3 Tissue Processor. In certain embodiments, the medium agitation is a stirring speed which one of ordinary skill in the art would consider equivalent to about 59-60 rpm on a HistoCore PELORIS 3 Tissue Processor (e.g., equivalent fluid speed, fluid mixing, and/or amount of energy introduce to the fluid). In certain embodiments, the medium agitation is an amount of agitation which one of ordinary skill in the art would consider equivalent to about 59-60 rpm on a HistoCore PELORIS 3 Tissue Processor (e.g., equivalent fluid speed, fluid mixing, and/or amount of energy introduce to the fluid).
Certain embodiments employ high agitation. For example, in certain embodiments, the high agitation is a stirring speed of about 79-80 rpm on a HistoCore PELORIS 3 Tissue Processor. In certain embodiments, the high agitation is a stirring speed of about 79-80 rpm on equipment equivalent to a HistoCore PELORIS 3 Tissue Processor. In certain embodiments, the high agitation is a stirring speed which one of ordinary skill in the art would consider equivalent to about 79-80 rpm on a HistoCore PELORIS 3 Tissue Processor (e.g., equivalent fluid speed, fluid mixing, and/or amount of energy introduce to the fluid). In certain embodiments, the high agitation is an amount of agitation which one of ordinary skill in the art would consider equivalent to about 79-80 rpm on a HistoCore PELORIS 3 Tissue Processor (e.g., equivalent fluid speed, fluid mixing, and/or amount of energy introduce to the fluid).
One aspect of the present disclosure is the step of contacting a reagent with the tissue sample. One of ordinary skill in the art will understand that there are multiple ways of contacting a reagent with a sample. For example, dipping a sample into a container of reagent or pouring or pumping a reagent into a container, chamber, or retort holding a sample. In certain embodiments, the step of contacting can be performed by a processor. For example, cassettes holding tissue samples are loaded into a basket that holds multiple cassettes, the basket is loaded into a retort which can hold multiple baskets, and reagent can be pumped into the retort to contact the samples from multiple stations at the corresponding processing step.
One aspect of the present disclosure is the step of removing a reagent from contact with the tissue sample, e.g., prior to the next step of contacting the tissue sample with the next reagent. Removing a reagent can be accomplished, for example, by pouring or draining away the reagent and/or pumping the reagent out of a chamber containing a sample. While it is desirable to remove as much reagent as possible, it is understood that some amount of reagent carryover is acceptable, expected, and accounted for in the process, and by “removing,” it is not necessary that all of the prior reagent be removed before the following step. For example, in certain embodiments where the tissue sample sits in a chamber or retort filled with a reagent, the reagent is pumped out and as much reagent as possible is drained off of the sample during a “drip time” before the next reagent is pumped in. In certain embodiments, a vacuum may be applied to aid in removing the reagent, for example during a paraffin step to remove residual xylene. It has been observed that using ActivFlo cassettes aid in less carryover which can translate to less reagent exchange time. While a wash step (such as using water or a wash buffer) may be employed between the twelve reagent steps, such wash steps are not necessary (some amount of reagent carry over is expected and accounted for) and increase the time, cost, and complexity of the process, generally without appreciable improvement.
Following the final step, the tissue sample can be further processed such as by embedding the tissue and/or used in staining protocols.
A 5-hour tissue processing protocol has been developed for processing tissue types, including breast, uterine, and gastrointestinal samples. In this example, the samples have a dimension of 20×20×4 mm. This protocol reduces total processing time when compared to other PELROIS 8-hour and 12-hour xylene factory protocols (e.g., HistoCore PELORIS 3 Premium Tissue Processing System User Manual, Melbourne), while also maintaining high quality tissue processing. In this example, testing was conducted using IP ActivFlo I cassettes on the HistoCore PELORIS 3 Tissue Processor to verify if the 5-hour xylene processing protocol could be installed and offered as a factory protocol in the future. Results of the testing conducted met the required acceptance criteria. The results of testing show that the quality of tissue processing derived from a 5-hour xylene protocol executed using a HistoCore PELORIS 3 Tissue Processor in combination with IP ActivFlo 1 cassettes met the acceptance criteria required of a factory protocol.
Acceptance Criteria Tested: HistoCore PELORIS 3 Tissue Processor passed a Verification of Installation Tissue Processing Run. Test set must achieve a minimum pass rate of 95% as specified in the PELORIS Tissue Processing Verification Test Plan, Melbourne.
Scoring System: Each sample was graded based on an assessment of 26 different parameters including both macroscopic and microscopic appearance (PELORIS Slide Evaluation Sheet, Melbourne). This allowed a detailed assessment to be made of the overall quality of tissue processing. The acceptance criteria were based on statistically relevant criteria. When analyzing results, any tissue sample that received a score above 80% in categories one (1) through six (6) in Table 1 was of high quality, and tissue samples below 50% were a failure.
Sample size: Based on a pass rate of 95% and a minimal acceptable value of 90% for the population, the sample size required for 80% power to achieve lower confidence level greater than the minimal acceptable value was 231 samples.
A sample size of 240 was used for this verification testing. No control samples were run. Testing was performed as per PELORIS Tissue Processing Verification Test Plan. Testing was conducted using a 110 V HistoCore PELORIS 3 Tissue Processor configured for xylene processing and three basket fill level.
Test Execution: Each of the IP ActivFlo Routine I cassettes was printed with a unique identifier detailing the tissue name and test sample number following the layout shown in Table 4.
The tissue samples were grossed and placed into cassettes and then into baskets as outlined in Table 5 and Table 6, with empty IP ActivFlo Routine I cassettes placed between each of the test cassettes to represent a full basket.
Each of the baskets were then loaded into the HistoCore PELORIS 3 Tissue Processor, which was filled with fresh reagents and prepped for testing as outlined in the HistoCore PELORIS 3 Premium Tissue Processing System User Manual, Melbourne. Verification of installation runs was completed and documented.
Four (4) test processing runs were conducted using the 5-hour xylene processing protocol, outlined in Attachment Table 7, with a Quick Clean Protocol executed between each test processing run.
The processing runs were performed using Retort A and Retort B alternatively to account for retort-to-retort variability, as outlined in Table 4.
After each processing run was completed, tissue samples were embedded. Blocks were trimmed free of excess paraffin and then faced until the tissue was accessible. After facing, each block was placed onto ice to be chilled and re-hydrated before being sectioned. Each block was sectioned by laying out a ribbon of at least 3-5 sections. Sections were mounted onto a pre-labeled slide and left to air dry overnight.
Prior to staining, all reagents on the HistoCore SPECTRA Stainer (LEICA BIOSYSTEMS, Richmond, IL) were changed and documented according to Table 8 and Table 9. Slides were stained using a Moderate Protocol and then coverslipped. These slides were then examined microscopically and scored.
Test Case Results: Following procedural steps followed in the HistoCore PELORIS 3 Premium Tissue Processing System User Manual, Melbourne, the Verification of installation runs passed, and the testing was able to proceed.
Slide scoring results followed the acceptance criteria outlined in the PELORIS Tissue Processing Verification Test Plan, Melbourne, and found 13 different slide and block combinations with subsections that scored less than 50%, which are individually considered failures. By calculating the number of failures against the sample size of 240 samples, the resulting pass rate was determined to be 95%, satisfying the acceptance criteria of the 95%.
Individual Tissue Type Scoring: Review of the data for the breast blocks individually, showed that 11 of the 13 highlighted blocks failed in the “Cutting” subcategory. In subsequent grading of the quality of the section, microscopic preservation and total score, blocks had a score above the 50% pass rate, demonstrating overall acceptability (PELORIS Tissue Processing Verification Test Plan, Melbourne).
Two blocks (B181 and B126) had scores of 0% for every category. These specific blocks were not able to be sectioned under the constraints of the written protocol, such as only allowing for chilling via an ice block and for a maximum of 15 minutes. As a result of not being able to obtain a full section, subsequent microscopic evaluation was not possible, which is documented in the subcategories having a score of 0%.
Tissue Type Averages: The averages for individual categories graded for each of the three (3) tissue types is summarized in Table 10, Table 11, and Table 12.
The slide scoring criteria document (PELORIS Tissue Processing Verification Test Plan, Melbourne) details that a score greater than 80% indicates that the quality of tissue processing is high, and a score of less than 50% is a failure. Comparing the averages between each of the three (3) tissue types, the total score is greater than 80% for each tissue type. Accordingly, the blocks and slides passed, and the quality of tissue processing assessed as high. Within each of the individual categories, none of the tissue types received a score of less than 50%, further demonstrating acceptability.
Slide Staining Procedure: When compiling data for analysis, it was discovered that 16 blocks were missed in the original testing. Those blocks were sectioned onto slides and placed into an oven for one hour at 60° C. for drying instead of being left to dry overnight. This deviation is acceptable because the baking method is not expected to impact the tissue processing quality. The slides were then stained and scored.
Processing Basket Layout: During the original testing that followed Table 5 and Table 6, three (s) baskets were loaded into each retort on two (2) for a total of four (4) tissue processing runs. It was discovered after processing that the basket number setting for reagent fill level on the HistoCore PELORIS 3 Tissue Processor was set to two (2) baskets instead of the three (3) that was required for testing. As a result, the 80 cassettes in the top basket in retorts A and B for each of the four (4) tissue processing runs were not exposed to an adequate volume of reagent and as a result had to be discarded. To account for the cassettes that had to be discarded, 80 cassettes were processed following the testing protocol as detailed above. To keep conditions consistent with original testing, empty IP ActivFlo I cassettes were placed in between test samples as well as the lower two processing baskets to mimic the original 160 sample size of cassettes. Following processing, the tissue in these 80 cassettes followed the same steps outlined in the protocol as the original 160 cassettes. This deviation is acceptable due to maintaining the same testing conditions and samples as per the test plan which did not impact the assessment of the 5-Hour tissue processing protocol.
Conclusion: The Examples demonstrate that the quality of processing of breast, uterine, and gastrointestinal samples using a 5-hour xylene protocol are satisfactory and with the same quality to replace prior, more time consuming protocols such as well-known 8 or 12 hours protocol.
The breadth and scope of the present disclosure should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.
This application claims priority to U.S. Provisional Application No. 63/459,483, filed Apr. 14, 2023, which is incorporated by reference herein in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63459483 | Apr 2023 | US |
