Improved method for frozen section of gastrointestinal tissue

Abstract

The invention discloses an improvement method of a gastrointestinal tissue frozen section. Aiming at the problems of long freezing time, easy formation of ice crystals, cell edema, many cracks and poor slice quality of a traditional method (standing and quick-freezing for 3 minutes at-20 DEG C to-30 DEG C after OCT embedding), the method comprises the following steps: pre-cooling a freezing head, trimming into a horizontal platform, placing gastrointestinal tissues in the center of the platform, limiting the shape by using a 3mm steel ring, and pressurizing and quickly freezing by using a freezing hammer (8-10 seconds) after OCT is dropwise added, and then directly slicing and staining. The method greatly shortens freezing time, reduces ice crystal damage, is clear in cell morphology and complete in section, improves diagnosis accuracy, is simple and convenient to operate, and is suitable for clinical rapid diagnosis requirements.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an improved method for frozen sections of gastrointestinal tissue. Background Technology

[0002] Intraoperative rapid frozen sectioning of gastrointestinal tissue is a crucial support for surgical decision-making, with its core objective being to maximize the timeliness of pathological slide preparation while ensuring diagnostic accuracy. Traditional frozen sectioning techniques typically involve directly encapsulating tissue samples with OCT embedding agents and statically freezing them in a constant-temperature environment of -20°C to -30°C via air convection. This process requires more than 3 minutes to allow the sample to fully solidify. During this period, free water molecules within the tissue cells continuously migrate and reorganize, inevitably forming large ice crystal structures. These ice crystals generate a mechanical expansion effect at the microscopic level, leading to cell membrane rupture, nuclear chromatin diffusion, and tearing of interstitial spaces, ultimately appearing as cavities, fissures, and localized edema artifacts in the slides. This damage is particularly pronounced in water-rich gastrointestinal mucosal biopsies (such as atrophic gastritis or adenoma samples), often causing false infiltration or distortion of glandular structures, directly affecting tumor boundary determination and differentiation assessment.

[0003] Existing technological improvements primarily focus on increasing the freezing rate. While liquid nitrogen jetting can accelerate freezing, the large temperature difference causes a surge in tissue fragility, leading to easy peeling or layered fragmentation during sectioning. Pressurized freezing devices use metal conductive plates for contact cooling; however, the lack of control over the shaping of the embedding medium causes the exposed tissue surface to easily adhere to the conductive plate, resulting in mechanical damage during peeling. More critically, traditional embedding processes generally suffer from uneven heat conduction pathways: as the OCT embedding medium solidifies layer by layer from the periphery to the core, the internal thermal gradient distribution of the sample becomes disordered, prolonging the effective freezing time (requiring waiting for the thickest area to fully solidify) and increasing the distance water migrates, creating a vicious cycle. Clinically, to avoid ice crystal damage, excessive trimming strategies are often adopted, leading to the loss of small nodular lesions (such as early cancer lesions with a diameter <3mm) during repeated trimming.

[0004] Equipment dependence is also a current technological bottleneck. Commercial rapid freezing instruments require customized cold source interfaces, making them difficult to popularize in primary hospitals. While fully automated embedding systems allow for standardized operation, they have poor adaptability to atypical tissues (such as the pedicle of tubular adenomas) and cannot solve the core problem of heat conduction efficiency. Studies (Glasgow Center for Pathology 2024) show that approximately 41% of defects in gastrointestinal frozen sections stem from a lack of morphological control of the tissue-embedding complex during the pre-freezing stage: uneven embedding block thickness leads to fluctuations in blade resistance, which is a major cause of section wrinkling and uneven thickness. Existing technologies alleviate this problem through empirical manual trimming, but manual operation is time-consuming and has low standardization, with a failure rate as high as 35% in acute bleeding or mucus-rich samples.

[0005] In summary, this invention addresses the core dilemma in gastrointestinal tissue frozen sectioning—the difficulty of simultaneously achieving freezing timeliness and cell morphology fidelity—by proposing a groundbreaking structured cryotransmission system. Through precise shaping of the pre-cooled metal interface, spatial constraints of the height-limited embedding frame, and three-dimensional synergy of directional pressure transmission, minimally invasive rapid freezing is achieved within an 8-10 second time window. Clinical validation shows a 76% reduction in key ice crystal damage indicators (number of cracks per unit area), and the slide preparation cycle is reduced to one-third of traditional methods. It provides a reliable technical foundation, particularly for the accurate assessment of small lesions (≤5mm) during surgery. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the existing technology and provide an improved method for frozen sections of gastrointestinal tissue, which improves diagnostic accuracy, is easy to operate, and is suitable for the needs of rapid clinical diagnosis.

[0007] The technical solution to achieve the objective of this invention is: an improved method for frozen sections of gastrointestinal tissue, characterized by comprising the following steps: (1) Pre-cooling and shaping of the freezing head: In the low-temperature working environment of the freezing machine or on the ice surface, place the clean metal freezing head flat in the freezing tank of the freezing machine, drip a sufficient amount of freezing embedding agent on its surface, and quickly transfer it back to the freezing tank until the embedding agent is completely solidified; after taking it out, use a pre-cooled blade or a trimmer to quickly rough trim the surface to form a horizontal and smooth platform. (2) Tissue placement and embedding: In the low-temperature working environment of the cryostat or on ice, place a gastrointestinal tissue sample of appropriate size and smaller than the inner diameter of the steel ring (12-18mm) quickly in the center of the platform; take a metal steel ring with an inner diameter that matches the tissue sample and a height of 2-3mm, coat the inner ring with a hydrophobic agent, and gently put it around the tissue block on the platform; quickly add sufficient freezing embedding agent into the steel ring to ensure that the tissue is completely submerged and the steel ring is filled; immediately place a metal freezing hammer pre-cooled to the working temperature of the cryostat vertically and stably on the surface of the embedding agent above the steel ring, apply directional pressure for 8-10 seconds until the embedding agent inside the steel ring is completely opaque and hardened and solidified; (3) Section preparation and staining: After confirming that the embedding agent has completely solidified, carefully remove the metal ring; load the cryostat onto the head holder of the cryostat, cut it into 4-6 μm thin sections on the cryostat, and attach them to the glass slide; fix with AAF fixative for 15 seconds, and then perform rapid HE staining. The rapid HE staining includes hematoxylin staining of the cell nucleus and eosin staining of the cytoplasm. The entire staining process shall not exceed 10 minutes.

[0008] As a further improvement, in step (1), the cryo-embedding agent is Sakura OCT cryo-embedding agent.

[0009] As a further improvement, in step (1), the pre-cooling to the working temperature of the freezer is -20℃.

[0010] As a further improvement, in step (1), the directional pressurization of the metal freezing hammer is based on the weight of the freezing hammer itself.

[0011] As a further improvement, in step (2), the metal ring is made of stainless steel and has a height of 3mm.

[0012] As a further improvement, in step (2), the hydrophobic agent is polytetrafluoroethylene, and the coating thickness is 0.1-0.2 mm.

[0013] As a further improvement, in step (3), the AAF fixative is composed of 10 ml of 40% formaldehyde, 85 ml of 95% ethanol and 5 ml of glacial acetic acid.

[0014] As a further improvement, in step (3), the temperature of the constant-temperature slicer is set to -20℃ to -30℃.

[0015] As a further improvement, in step (3), the hematoxylin staining time for rapid HE staining is 90s-120s, and the eosin staining time is 10-15s.

[0016] After adopting the above technical solution, the present invention has the following positive effects: Significantly improved freezing efficiency: This invention combines pre-cooling and shaping of the freezing head, positioning with a 3mm height-limiting steel ring, and directional pressurization with a metal freezing hammer. By utilizing the large heat capacity of the freezing hammer and the thermal conductivity of metal, the effective freezing and solidification time of tissue and embedding agent is shortened to 8-10 seconds (compared to more than 3 minutes in traditional methods). The freezing efficiency is improved by orders of magnitude, solving the pain points of traditional freezing methods that are time-consuming and cannot meet the needs of rapid intraoperative diagnosis.

[0017] Effective inhibition of ice crystal damage: In the above technical solution, the significant reduction in freezing time reduces the migration time of free water in the tissue and lowers the probability of large ice crystal formation; at the same time, the shaping effect of the 3mm height-limiting steel ring makes the tissue and embedding agent form a uniform thin block, further optimizing heat dissipation efficiency, thereby inhibiting problems such as cell edema and morphological damage caused by ice crystal compression and maintaining the integrity of cell structure.

[0018] Significantly improved section quality: Due to reduced ice crystal damage, sections prepared by this invention are less prone to defects such as voids, cracks, or tissue fragmentation. The continuity, smoothness, and integrity of the sections are significantly improved. Cell morphology is clear and nuclear staining is uniform (e.g., in the modified section, the cell morphology of tissues such as colonic leiomyomas and severe gastric antral atrophy with intestinal metaplasia is intact, with no obvious edema or cracks), providing pathologists with a clearer field of view, which helps to improve the accuracy and reliability of diagnosis and meet the clinical demand for "precision" frozen sections.

[0019] Simplified operation process: This invention simplifies the "waiting for tissue to freeze naturally" step in traditional freezing methods by using a pre-cooling freezing head, a steel ring for height limitation, and a freezing hammer for pressure. No additional waiting time is required before sectioning, making the operation more convenient and reducing the workload of pathology technicians. Attached Figure Description

[0020] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein... Figure 1 This is a schematic diagram of the core steps of the present invention (pre-cooling and trimming, 3mm steel ring positioning, and freezing hammer pressurization).

[0021] Figure 2 Microscopic comparison of frozen tissue sections prepared by the present invention and the traditional air freezing method (left: improved method, right: traditional method).

[0022] Figure 3 This is a microscopic demonstration of the improved frozen section method of the present invention. Detailed Implementation

[0023] The embodiments of the present invention are described in detail below. It should be noted that these embodiments are descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0024] Unless otherwise specified, the raw materials used in this invention are all conventional commercially available products; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0025] Specifically, the relevant verification embodiments in this invention are as follows: Example 1

[0026] 1. Preparation of materials and equipment 1.1 Experimental Specimens Forty frozen specimens of clinical endoscopic gastrointestinal biopsies collected from the Hongxing Hospital of the 13th Division of Xinjiang Production and Construction Corps between June and July 2025 were selected. Among them, 23 were male and 17 were female, aged 20–85 years (median age 54 years). Final pathological diagnoses included: chronic gastritis with active inflammation in 9 cases, chronic atrophic gastritis in 12 cases, high-grade intraepithelial neoplasia of the gastric mucosa in 2 cases, gastric polyps in 8 cases, colonic adenomas in 6 cases, colon cancer in 1 case, and gastric cancer in 2 cases. All specimens were freshly collected gastrointestinal tissues, unfixed or only briefly (≤30 minutes) refrigerated (4℃).

[0027] 1.2 Experimental Equipment Cryosectioning system: Leica CM 1950S cryostat (operating temperature set to -20℃ to -30℃); Auxiliary equipment: Fully automatic frozen section staining machine (domestic), pre-cooling trimming knife (stainless steel, pre-cooled to -20℃ in the freezer); Core improvement tools: 3mm high metal steel ring (inner diameter adapted to the freezing head platform, made of stainless steel, with the inner ring pre-coated with polytetrafluoroethylene hydrophobic agent), metal freezing hammer (smooth bottom surface, pre-cooled to -20℃ in the freezing machine).

[0028] 1.3 Reagents Embedding and fixation: Sakura OCT cryo-embedding medium, AAF fixative (10ml of 40% formaldehyde + 85ml of 95% ethanol + 5ml of glacial acetic acid, prepared fresh for immediate use); Staining and mounting: Hematoxylin staining solution, eosin staining solution, blueing solution (1% ammonia, pH=8.5), clearing solution (xylene), and neutral resin mounting medium from Zhuhai Beso Company.

[0029] 2. Implementation Steps (The following steps are all performed within the low-temperature working chamber (-20℃ to -30℃) of the Leica CM 1950S cryostat to avoid tissue warming.) Figure 1 As shown.

[0030] 2.1 Pre-cooling and shaping of the freezing head Take a clean metal cryohead (50mm in diameter), place it flat in the freezing tank (-20℃) of the freezer, and drop 2ml of Sakura OCT freezing embedding agent onto its surface to ensure that it covers the entire surface of the cryohead. Place the cryoprobe in the freezing tank for 3 minutes until the Sakura OCT cryo-embedding agent is completely solidified (into a milky white solid), forming the bottom embedding layer.

[0031] 2.2 Rough finishing and leveling treatment Using a stainless steel trimmer pre-cooled to -20℃, quickly scrape off the solidified Sakura OCT freezing embedding agent layer on the surface of the freezing head until a horizontal, smooth platform is exposed (error ≤ 0.5mm). The scraping process must be completed within 10 seconds to prevent the temperature of the freezing head from rising again (you can check this by touching the edge of the freezing head; if it feels warm, put it back into the freezing tank for 1 minute to pre-cool).

[0032] 2.3 Organization Placement and Steel Ring Height Limit Take a fresh gastrointestinal tissue specimen (size ≤10mm×10mm×5mm), quickly trim it into a slice with a thickness ≤3mm (matching the height of the steel ring), and place it in the center of the cryoprobe platform; Gently place a 3mm high metal ring (15mm inner diameter) pre-coated with polytetrafluoroethylene hydrophobic agent around the tissue, ensuring that the bottom of the ring is completely in contact with the cryoprobe platform and that the top of the tissue does not exceed the height of the ring (this can be adjusted by visual inspection or by gently pressing with pre-cooled tweezers).

[0033] 2.4 Secondary injection and directional pressure freezing Add 1 ml of Sakura OCT cryoemulation medium into the steel ring, ensuring that the tissue is completely submerged and the steel ring is filled (avoid air bubbles; if air bubbles are present, puncture them with pre-cooled forceps). Immediately take a metal freezing hammer that has been pre-cooled to -20°C and press it vertically onto the surface of the Sakura OCT freezing embedding agent above the steel ring, maintaining pressure for 8~10 seconds; After 8 seconds, gently shake the freezing hammer upwards. If the freezing hammer separates from the surface of the Sakura OCT cryo-embedding agent and the Sakura OCT cryo-embedding agent is in a completely opaque solid state, stop pressurizing. If it is not completely solidified, extend the pressurizing time by 2 to 3 seconds.

[0034] 2.5 Sectioning and Staining Carefully remove the underwire (use pre-cooled tweezers to gently pry the edge of the underwire; the underwire can be quickly removed by utilizing the anti-stick properties of the hydrophobic agent). Load the cryoprobe onto the cryostat holder, adjust the section thickness to 5 μm (normal thickness for gastrointestinal tissue), and start the cryostat for continuous sectioning; Use a pre-cooled glass slide (-20℃) to receive the slide, and attach the slide to the center of the glass slide (avoid wrinkles). Immerse the slide in AAF fixative for 15 seconds (fixation time error ≤ 2 seconds), then rinse twice with distilled water (3 seconds each time). Immerse the cells in hematoxylin staining solution for 90-120 seconds, rinse once with distilled water (2 seconds), and then soak in blueing solution for 5 seconds (to make the cell nuclei dark blue). Immerse the slide in eosin staining solution for 10 seconds, rinse once with distilled water (2 seconds), dehydrate with 75%, 85%, and 95% ethanol (10 seconds), then anhydrous ethanol (10 seconds), clear with xylene (10 seconds), and finally mount with neutral resin (the whole process takes about 10 minutes).

[0035] 3. Comparison Experiment with Traditional Methods To verify the effectiveness of the improved method, 10 gastrointestinal tissue specimens from the same source were selected and processed using the traditional frozen sectioning method, as follows: Tissue blocks were embedded in OCT embedding medium (a water-soluble mixture) and placed in a cryostat (at -20°C to -30°C) for 3 minutes to freeze and harden the tissue. The sections were 4-6 μm thick and fixed with methanol / ethanol fixative (1:1) for 30 seconds. Hematoxylin staining for 4 minutes, eosin staining for 20 seconds, and dehydration and clearing process for a total of 15 minutes (approximately 30 minutes in total).

[0036] 4. Effect Verification and Results 4.1 Comparison of Freezing Aging Time Improved method: The time from placement to complete freezing of tissue is 8-15 seconds (average 10 seconds); Traditional method: tissue freezing time is 3 minutes (180 seconds), improved method improves efficiency by 18 to 22 times.

[0037] 4.2 Ice Crystal Damage Assessment Sections of gastric antrum tissue (severe atrophy with intestinal metaplasia) processed using both the modified and traditional methods were observed under an optical microscope (×400x): Improved sectioning method: Cells are morphologically intact, without obvious ice crystal cavities (cavity area ≤5%), and cell edema rate (proportion of cells with an increase in cell volume ≥20%) ≤10%; (see Figure 2 Left) Traditional method sectioning: ice crystal voids and fissures ≥30% area, cell edema rate ≥40% (see...) Figure 2 right).

[0038] 4.3 Concordance rate between slide quality and diagnosis Of the 40 sections prepared using the improved method, 38 (95%) reached the "excellent" grade (standard: continuous sections, no cracks, clear cell morphology, and uniform nuclear staining), and 2 (5%) were "qualified" (slight fragmentation due to the brittle texture of the tissue itself, but this did not affect the diagnosis). Of the 10 slides prepared using traditional methods, only 3 (30%) reached the "excellent" grade, 5 (50%) were "qualified", and 2 (20%) were "unqualified" (≥2 clefts or unclear cell morphology, making diagnosis impossible). The diagnostic accuracy rate of the improved method (compared with postoperative paraffin sections) was 98% (39 / 40), while that of the traditional method was 85% (17 / 20) (data from the Department of Pathology, Hongxing Hospital, July 2025).

[0039] 5. Display of typical specimens 5.1 Colonic leiomyoma specimen Specimen details: Descending colon biopsy tissue, measuring 8mm × 6mm × 3mm, with a relatively soft texture; Improved processing method: steel ring height 3mm, pressure time 8 seconds, slice thickness 5μm; Results: The leiomyomas in the sections were clearly morphologically distinct (spindle cells arranged neatly), with uniform nuclear staining (neither dark nor light staining), and no clefts or cavities (see [link to original text]). Figure 3 Left); Diagnosis: Consistent with the postoperative paraffin section diagnosis (colonic leiomyoma).

[0040] 5.2 Specimen of severe gastric antral atrophy with intestinal metaplasia Specimen details: Gastric antrum biopsy tissue, measuring 10mm × 8mm × 4mm, with a relatively brittle texture; Improved treatment method: Trim tissue thickness to 3mm, steel ring height to 3mm, and apply pressure for 10 seconds; Results: Gastric gland atrophy (reduced number) and intestinal metaplastic cells (goblet cells) were clearly visible in the sections, with no cellular edema (see [link to article]). Figure 3 right); Diagnosis: Consistent with the postoperative paraffin section diagnosis (severe gastric antral atrophy with intestinal metaplasia).

[0041] 6. Conclusion This specific embodiment details an improved method for frozen sections of gastrointestinal tissue. Through a core process of "pre-cooling and shaping - rough trimming and smoothing - height restriction with a steel ring - pressure freezing," the tissue freezing time is reduced from 3 minutes in the traditional method to 8-15 seconds, significantly inhibiting ice crystal formation and improving section quality. This method is simple to operate, low in cost (the steel ring and freezing hammer are reusable), and suitable for widespread application in clinical pathology departments.

[0042] The working principle of this invention is as follows: A flat embedding platform is constructed using a pre-cooled metal cryoprobe. A height-limited steel ring (critical dimension 3mm) coated with a hydrophobic agent tightly encloses fragile gastrointestinal biopsy tissue. Immediately after the secondary injection of embedding agent, a pre-cooled metal cryo-hammer is applied for directional pressure conduction. This steel ring-cryo-hammer synergistic system prevents tissue deformation under pressure through physical limitation and utilizes the high thermal conductivity of the cryo-hammer to achieve efficient and directional heat dissipation within the tissue, enabling the sample to achieve deep homogeneous freezing within 8-10 seconds. Its core breakthrough lies in the fact that the height-limited steel ring accelerates the freezing path while maintaining the original morphology of the biopsy tissue, while the cryo-hammer inhibits ice crystal formation through forced heat conduction. Together, they overcome the problem of tissue fragility caused by traditional direct pressure, ultimately achieving complete preservation of cell structure and rapid, stable slide preparation.

[0043] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An improved method for frozen sections of gastrointestinal tissue, characterized in that, Includes the following steps: (1) Pre-cooling and shaping of the freezing head: In the low-temperature working environment of the freezing machine or on the ice surface, place the clean metal freezing head flat in the freezing tank of the freezing machine, drip a sufficient amount of freezing embedding agent on its surface, and quickly transfer it back to the freezing tank until the embedding agent is completely solidified; after taking it out, use a pre-cooled blade or a trimmer to quickly rough trim the surface to form a horizontal and smooth platform. (2) Tissue placement and embedding: In the low-temperature working environment of the cryostat or on ice, place a gastrointestinal tissue sample of appropriate size and smaller than the inner diameter of the steel ring (12-18mm) quickly in the center of the platform; take a metal steel ring with an inner diameter that matches the tissue sample and a height of 2-3mm, coat the inner ring with a hydrophobic agent, and gently put it around the tissue block on the platform; quickly add sufficient freezing embedding agent into the steel ring to ensure that the tissue is completely submerged and the steel ring is filled; immediately place a metal freezing hammer pre-cooled to the working temperature of the cryostat vertically and stably on the surface of the embedding agent above the steel ring, apply directional pressure for 8-10 seconds until the embedding agent inside the steel ring is completely opaque and hardened and solidified; (3) Section preparation and staining: After confirming that the embedding agent has completely solidified, carefully remove the metal ring; load the cryostat onto the head holder of the cryostat, cut it into 4-6 μm thin sections on the cryostat, and attach them to the glass slide; fix with AAF fixative for 15 seconds, and then perform rapid HE staining. The rapid HE staining includes hematoxylin staining of the cell nucleus and eosin staining of the cytoplasm. The entire staining process shall not exceed 10 minutes.

2. The improved method according to claim 1, characterized in that, In step (1), the cryo-embedding agent is Sakura OCT cryo-embedding agent.

3. The improved method according to claim 1, characterized in that, In step (1), the pre-cooling to the working temperature of the freezer is -20℃.

4. The improved method according to claim 1, characterized in that, In step (1), the directional pressurization of the metal freezing hammer is based on the weight of the freezing hammer itself.

5. The improved method according to claim 1, characterized in that, In step (2), the metal ring is made of stainless steel and has a height of 3mm.

6. The improved method according to claim 1, characterized in that, In step (2), the hydrophobic agent is polytetrafluoroethylene, and the coating thickness is 0.1-0.2 mm.

7. The improved method according to claim 1, characterized in that, In step (3), the AAF fixative is composed of 10 ml of 40% formaldehyde, 85 ml of 95% ethanol and 5 ml of glacial acetic acid.

8. The improved method according to claim 1, characterized in that, In step (3), the temperature of the constant-temperature slicer is set to -20℃ to -30℃.

9. The improved method according to claim 1, characterized in that, In step (3), the hematoxylin staining time for rapid HE staining is 90s-120s, and the eosin staining time is 10-15s.

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