A liquid-based cytology preservative composition free of formalin and method thereof

Abstract

The present invention relates to a liquid-based cytology (LBC) preservative composition, offering a formalin-free and PEG-free solution for preserving biological samples The composition comprises 25% to 40% w / v ethanol as a preservative and antifungal agent, 0.25% w / v Polysorbate-80 as a non-ionic detergent stabilizing surface receptors, 0.1M w / v citric acid for enhancing DNA / RNA preservation, 0.2% w / v 2-Chloroacetamide for antimicrobial activity, 0.45% w / v sodium chloride for cellular integrity, and 0.5% w / v sodium hydroxide for maintaining pH at 6.4 ± 0.4. The solution is adjusted to 100% w / v using deionized water. This preservative is versatile, compatible with gynecological, non- gynecological, FNAC, and oral cytology samples, and preserves cellular morphology and molecular integrity for advanced studies, including immunocytochemistry, HPV DNA analysis, and gene expression. Its formalin-free nature eliminates carcinogenic risks, offering a safer and more effective alternative for medical and research applications. The composition ensures long-term stability and usability.

Description

[0001] A LIQUID-BASED CYTOLOGY PRESERVATIVE COMPOSITION FREE OF FORMALIN AND METHOD THEREOF EARLIEST PRIORITY DATE:

[0002] This Application claims priority from a Provisional patent application filed in India having Patent Application No. 202441096604, filed on December 06, 2024, and titled “A FORMALIN-FREE LIQUID-BASED CYTOLOGY PRESERVATIVE AND A METHOD THEREOF”.

[0003] TECHNICAL FIELD

[0004] The present invention pertains to the field of biological sample preservation and cytology, specifically to the development of a liquid-based cytology (LBC) preservative composition. The invention focuses on providing a formalin-free and PEG-free preservative that ensures the effective preservation of cellular morphology, molecular integrity, and surface receptors for various medical and research applications.

[0005] BACKGROUND OF THE INVENTION

[0006] Liquid-based cytology (LBC) has significantly advanced cytological analysis by providing better cell preservation and enhanced diagnostic accuracy compared to traditional smear techniques. In this process, preservatives play a vital role in maintaining cellular morphology and molecular integrity, which are critical for cytological and molecular analyses. However, conventional preservatives often contain formalin, a chemical with known safety and performance limitations, which has prompted the need for alternative solutions.

[0007] Formalin-based preservatives are widely used in LBC due to their efficacy in preserving cellular structures, hardening cellular components, and stabilizing samples for long-term storage. They are compatible with routine staining methods and basic cytological analyses. Additionally, some preservatives incorporate polyethylene glycol (PEG) to stabilize surface receptors. Despite these advantages, formalin-based preservatives are unsuitable for advanced molecular diagnostic methods, such as DNA, RNA, and immunocytochemistry analyses, due to inherent limitations.

[0008] One major drawback of formalin-based solutions is their health hazard. Formalin is a potent carcinogen, and prolonged exposure poses severe risks to laboratory personnel, including respiratory and skin irritation. Additionally, formalin causes fragmentation of genetic material such as DNA and RNA, compromising its utility in molecular studies like gene expression and HPV DNA analysis. Moreover, formalin and PEG-based solutions can interfere with smear preparation and cytological staining, leading to inaccuracies in diagnostic outcomes. PEG, often used as an emulsifier, can destabilize surface receptors, making preserved samples less suitable for immunocytochemistry. Furthermore, the applicability of these preservatives is often limited to specific sample types, restricting their versatility in cytological practices.

[0009] These limitations underscore the urgent need for a formalin-free, multipurpose preservative that ensures safety and enhances diagnostic accuracy. A formalin-free solution eliminates the carcinogenic risks associated with formalin, offering a safer alternative for laboratory personnel and patients. Such a preservative would maintain the molecular integrity of genetic material, preventing DNA and RNA degradation and enabling accurate molecular analyses. Additionally, a PEG-free formula preserves surface receptor stability, ensuring reliable results in receptor-based studies like immunocytochemistry. A versatile preservative capable of handling diverse sample types — such as gynecological, non-gynecological, FNAC, and oral cytology — would simplify workflows and broaden its applicability in clinical and research settings.

[0010] By addressing the limitations of formalin-based solutions, a formalin-free preservative not only ensures safety but also aligns with the evolving needs of modem diagnostic and research methodologies, improving cytological and molecular diagnostic outcomes across various applications.

[0011] SUMMARY OF THE INVENTION

[0012] In the light of the disadvantages mentioned in the previous section, the following summary is provided to facilitate an understanding of some of the innovative features unique to the present invention and is not intended to be a full description. A full appreciation of the various aspects of the invention can be gained by taking the entire specification as a whole. According to an embodiment of the present invention, a liquid-based cytology preservative composition free of formalin is provided, comprising 25% to 40% w / v ethanol, which acts as a cell preservative and antifungal agent; 0.25% w / v Polysorbate-80, a non-ionic detergent that stabilizes surface receptors and inactivates viruses; 0.1M w / v citric acid, which enhances DNA and RNA preservation and reduces degradation; 0.2% w / v 2-Chloroacetamide, which provides antimicrobial activity; 0.45% w / v sodium chloride for maintaining cellular integrity; 0.5% w / v sodium hydroxide for maintaining pH balance; and deionized water to adjust the composition to a final concentration of 100% w / v. The pH of the composition is maintained at 6.4 ± 0.4 to ensure optimal stability and preservation of cellular morphology and molecular integrity.

[0013] According to another embodiment, the composition is formalin-free and PEG-free, thereby eliminating cytological staining interference, cellular hardening, and gene fragmentation associated with formalin-based preservatives. This formulation ensures compatibility with multiple sample types, including but not limited to gynecological cytology samples, non-gynecological cytology samples, fine needle aspiration cytology (FNAC) samples, and oral cytology samples.

[0014] According to yet another embodiment, the preservative composition maintains surface receptor integrity, enabling advanced testing methodologies such as immunocytochemistry, HPV DNA analysis, mRNA studies, and gene expression analysis. The composition ensures the preservation of molecular and cellular components without disruption, making it suitable for long-term storage and diagnostic accuracy.

[0015] According to an additional embodiment, the method for preparing the liquid-based cytology preservative composition involves mixing 25% to 40% w / v ethanol with deionized water, followed by the addition of 0.25% w / v Polysorbate-80, 0.1M w / v citric acid, and 0.2% w / v 2-Chloroacetamide. Subsequently, 0.45% w / v sodium chloride and 0.5% w / v sodium hydroxide are dissolved in the mixture to maintain cellular integrity and pH balance. The final volume is adjusted with deionized water to achieve a 100% w / v concentration, with the pH of the solution maintained at 6.4 ± 0.4.

[0016] According to a further embodiment, the composition is utilized to preserve biological samples by submerging the sample in the solution for a duration sufficient to maintain cellular morphology and molecular integrity. The preserved sample can then be used for cytological analysis, immunocytochemistry, and molecular studies, including DNA and RNA analyses. According to an additional embodiment of the present invention, the liquid-based cytology preservative composition replaces formalin-based solutions in medical and research settings, offering a safer, non-carcinogenic alternative that enhances diagnostic accuracy and ensures reliable results in advanced molecular and cytological studies.

[0017] Additional features and advantages of various embodiments will be set forth in part in the description that follows, and in part will be apparent from the description, or may be learned by practice of various embodiments. The objectives and other advantages of various embodiments will be realized and attained by means of the elements and combinations particularly pointed out in the description and appended claims.

[0018] BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

[0019] The detailed description is provided with reference to the accompanying figures. These and other features and advantages of the present invention will be readily appreciated, as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings wherein:

[0020] Fig. 1 is a flowchart illustrating a method for preparing a liquid-based cytology preservative composition free of formalin according to an embodiment of the present invention.

[0021] DETAILED DESCRIPTION

[0022] For the purpose of promoting an understanding of the principles of the disclosure, reference will now be made to the embodiment illustrated in the figures and specific language will be used to describe them. It will nevertheless be understood that no limitation of the scope of the disclosure is thereby intended. Such alterations and further modifications in the illustrated system, and such further applications of the principles of the disclosure as would normally occur to those skilled in the art are to be construed as being within the scope of the present disclosure.

[0023] The terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process or method that comprises a list of steps does not include only those steps but may include other steps not expressly listed or inherent to such a process or method. Similarly, one or more devices or sub-systems or elements or structures or components preceded by "comprises... a" does not, without more constraints, preclude the existence of other devices, sub-systems, elements, structures, components, additional devices, additional sub-systems, additional elements, additional structures, or additional components. Appearances of the phrase "in an embodiment", "in another embodiment" and similar language throughout this specification may, but not necessarily do, all refer to the same embodiment.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. The system, methods, and examples provided herein are only illustrative and not intended to be limiting.

[0025] In the following specification and the claims, reference will be made to a number of terms, which shall be defined to have the following meanings. The singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise.

[0026] According to an embodiment of the present invention, a liquid-based cytology preservative composition free of formalin is disclosed, which effectively preserves biological samples while eliminating the use of formalin, a known carcinogen. The composition comprises 25% to 40% w / v ethanol as a cell preservative and antifungal agent, 0.25% w / v Polysorbate-80 as a non-ionic detergent for stabilizing surface receptors and inactivating viruses, and 0.1M w / v citric acid to enhance the stability of DNA and RNA, reducing degradation over time. Additionally, the composition includes 0.2% w / v 2-Chloroacetamide as an antimicrobial agent, 0.45% w / v sodium chloride to maintain cellular integrity, and 0.5% w / v sodium hydroxide to stabilize the pH of the solution at 6.4 ± 0.4. Deionized water is used to adjust the formulation to a final concentration of 100% w / v.

[0027] According to another embodiment, the invention provides a versatile preservative that is suitable for multiple sample types, including gynecological cytology samples, non-gynecological cytology samples, fine needle aspiration cytology (FNAC) samples, and oral cytology samples. This universal applicability simplifies laboratory workflows and reduces the need for multiple preservatives, making it a cost-effective and efficient solution for a variety of diagnostic and research needs.

[0028] According to yet another embodiment, the preservative composition is formalin-free and PEG-free, overcoming critical drawbacks of conventional preservatives. Formalin-based solutions often cause cytological staining interference, hardening of cellular components, and fragmentation of genetic material such as DNA and RNA. Similarly, PEG disrupts surface receptor stability, impairing its use in immunocytochemistry. By eliminating these components, the present invention ensures the preservation of cellular morphology and molecular integrity without these adverse effects, thereby enhancing the reliability of downstream molecular analyses.

[0029] According to an additional embodiment, the composition preserves surface receptors without disruption, enabling advanced testing methodologies. These include immunocytochemistry, HPV DNA analysis, mRNA studies, and gene expression analysis, where the integrity of cellular and molecular components is critical. The absence of formalin and PEG ensures that receptors and genetic material remain intact, preventing loss of diagnostic and analytical precision.

[0030] Fig. 1 is a flowchart illustrating a method for preparing a liquid-based cytology preservative composition free of formalin according to an embodiment of the present invention. The method of preparing the liquid-based cytology preservative involves a stepwise mixing of the components to ensure homogeneity and stability. At step 102, 25% to 40% w / v ethanol is first mixed with deionized water, followed by the addition of 0.25% w / v Polysorbate-80 to act as a detergent at step 104. At step 106, 0.1M w / v Citric acid is added to enhance the stability of nucleic acids. Next at step 108, 0.2% w / v 2-Chloroacetamide is incorporated as an antimicrobial agent. At step 110, 0.45% w / v Sodium chloride and 0.5% w / v sodium hydroxide are dissolved in the mixture to maintain cellular integrity and pH stability. At step 112, the final solution is adjusted to 100% w / v with deionized water. Finally at step 114, the pH is fine-tuned to 6.4 ± 0.4 to optimize preservation conditions.

[0031] According to another embodiment, the preservative composition is used to preserve biological samples by submerging them in the solution for a sufficient duration to maintain cellular morphology and molecular integrity. Preserved samples remain stable for long-term storage and are suitable for a range of cytological and molecular studies. This includes cytological smear preparations, immunocytochemistry, and advanced molecular analyses such as DNA and RNA studies.

[0032] According to an additional embodiment of the present invention, this preservative composition addresses the critical need for safer and more effective alternatives to formalinbased solutions. By providing a formalin-free and PEG-free composition, the invention significantly reduces health risks to laboratory personnel and patients while ensuring compatibility with evolving diagnostic and research methodologies. The invention ensures superior preservation quality for various biological samples, contributing to improved diagnostic accuracy and research outcomes.

[0033] WORKING EXAMPLE OF THE PRESENT INVENTION

[0034] Preparation of the Liquid-Based Cytology Preservative Composition

[0035] Step 1: Preparation of Base Solution

[0036] A base solution was prepared by dissolving 30% w / v ethanol in deionized water. Ethanol was chosen for its dual role as a preservative and antifungal agent, ensuring long-term stability and microbial protection of biological samples.

[0037] Step 2: Addition of Polysorbate-80

[0038] To the ethanol solution, 0.25% w / v Polysorbate-80 was added while stirring. Polysorbate-80, a non-ionic detergent, stabilizes surface receptors on preserved cells and inactivates viruses. The mixture was stirred until completely dissolved.

[0039] Step 3: Incorporation of Citric Acid

[0040] 0.1M w / v citric acid was added to the solution to enhance the stability of DNA and RNA in preserved samples. Citric acid also reduces oxidative degradation of genetic material, critical for molecular analyses.

[0041] Step 4: Addition of Antimicrobial Agent

[0042] 0.2% w / v 2-Chloroacetamide was incorporated as an antimicrobial agent. This compound ensures microbial inhibition during the storage of biological samples.

[0043] Step 5: Balancing Ionic Strength

[0044] 0.45% w / v sodium chloride was dissolved in the mixture to maintain cellular integrity by balancing the ionic strength within the solution.

[0045] Step 6: pH Stabilization

[0046] 0.5% w / v sodium hydroxide was added to adjust the pH of the solution to 6.4 ± 0.4, which is optimal for cellular morphology preservation and molecular stability. The pH was checked using a calibrated pH meter.

[0047] Step 7: Final Adjustments

[0048] The total volume of the solution was adjusted to 100% w / v with deionized water, ensuring all components were fully dissolved. The mixture was filtered through a 0.22-micron filter to ensure sterility and stored in sterile containers for use.

[0049] EXPERIMENTAL STUDY DETAILS

[0050] 1. In-Use Stability Study of the Preservative Composition

[0051] An in-use stability study was conducted to demonstrate the ability of the liquid-based cytology preservative composition of the present invention to maintain its performance characteristics over a defined time interval after the first opening of the container.

[0052] Objective: The objective of the study was to determine the in-use stability of the preservative composition. The study aimed to verify that the performance of the composition for preserving cervical samples remains stable for at least four weeks when stored at room temperature (30±5°C) and for five weeks under refrigeration (15±5°C).

[0053] Methodology: The study was designed to assess the preservative capabilities of the composition at different time intervals and storage conditions.

[0054] • Specimen Type: Cervical samples collected using a cyto-brush were used for the study.

[0055] • Batch Selection: A minimum of two pilot-scale batches of the preservative composition were subjected to the test. To ensure robustness, at least one of the batches selected was towards the end of its established shelflife.

[0056] • Sample Preparation and Storage: A total of five freshly collected cervical samples were placed in vials containing 14 ml of the preservative composition. These five preserved samples were then split into two groups for storage under different conditions:

[0057] o Group I: Stored at room temperature (30±5°C).

[0058] o Group II: Stored under refrigeration (15±5°C).

[0059] DAY GROUP I GROUP II TOTAL SAMPLES

[0060] (30° ± 5°) (15° ± 5°)

[0061] 0thDAY RTGP1 RFGP1 5 + 5 7THDAY RTGP2 RFGP2 5 + 5 14THDAY RTGP3 RFGP3 5 + 5 21STDAY RTGP4 RFGP4 5 + 5

[0062]

[0063] 28THDAY RTGP5 RFGP5 5 + 5 | 35THDAY | RTGP6 | RFGP6 | 5 + 5 |

[0064]

[0065] • Test Procedure and Schedule: The stability of the preserved samples in both groups was analyzed at regular intervals over five weeks. Testing was performed on Day 0, Day 7, Day 14, Day 21, Day 28, and Day 35. At each interval, the sample containers were opened, and a 2 ml aliquot was taken from each sample in Group I and Group II to prepare cytological smears according to a standard liquid-based cytology procedure.

[0066] • Evaluation Parameters and Acceptance Criteria: The resulting smears were analyzed based on standard cytological evaluation parameters, including sample adequacy, the presence of cellular components of the transformation zone, reactive cellular changes, infective organisms, and abnormalities in squamous or glandular cells. All findings were reported and tabulated according to " The Bethesda System" for reporting cervical cytology.

[0067] Results: The performance of the preservative composition was evaluated at each time point by assessing the morphological integrity of the preserved cells.

[0068] • Group I (Room Temperature): The samples stored at room temperature (30±5°C) remained stable for a period of four weeks (28 days). The cellular morphology and diagnostic characteristics were well-preserved and comparable to the results from Day 0. Some degradation in sample quality was noted at the end of the fifth week (Day 35).

[0069] • Group II (Refrigeration): The samples stored under refrigeration (15±5°C) demonstrated excellent preservation for the entire five-week (35 -day) duration of the study. The samples showed good cellular yield and well-preserved morphology throughout the testing period.

[0070] • Note: Abbreviation

[0071] AD - Adequate A- Absent P-Present Cell Comp - Cellular component Tr - Transformation Reac - Reactive Sq

[0072] Squamous mal - Malignant Report format - The Bethesda System SPECIMEN TYPE Eziprep LBC

[0073] SITE OF SPECIMEN Ecto & Endo Cervix

[0074] VDNA / LBINU001

[0075] REPORT NO Dated 02 / 3 / 2021

[0076] Day Sample

[0077] RTGP1

[0078] SAMPLE 1 AD P P P A A A OTHDay SAMPLE 2 AD P P P A A A 30 ± 5° C SAMPLE 3 AD P A A A A A SAMPLE 4 AD P P P A A A SAMPLE 5 AD P A A A A A RFGP1

[0079] SAMPLE 1 AD P P P A A A OTHDay SAMPLE 2 AD P P P A A A 15 ± 5° C SAMPLE 3 A Ad Ad AequacydDequacyequacy P A A A A A SAMPLE 4 AD P P P A A A

[0080]

[0081] SAMPLE 5 AD C Cll T C Cll T C Ceomprll Teompreompr P A A A A A Z Z Zoneoneone

[0082] Table 1: 0thDay Observation

[0083] C Rll C Rll C Rlleaceeaceeace...

[0084] Ch Ch Cangeshangesanges

[0085] SPECIMEN TYPE Eziprep LBC

[0086] SITE OF SPECIMEN Ecto & Endo Cervix fi Itnecve

[0087] f O Inecrg.

[0088] VDNA / LBINU002 f O Inecrg Oi.rgansm REPORT NO Dated 09 / 3 / 2021

[0089] Day Sample

[0090] S Aiblt S Abl S Ablqnormayqnormaqnorma...

[0091] RTGP2

[0092] SAMPLE 1 AD P P P A A A Gldl Gldanua 7 SAMPL 2 Gldran THDay E AD P P P A an Aibltblbl anormay A anorma anorma 30 ± 5° C SAMPLE 3 AD P A A A A A SAMPLE 4 AD P P P A A Ohlt Ohl Ohlter matermaerma A SAMPLE 5 AD P A A A A Alll neopasm neopasm neopasm RFGP2

[0093] SAMPLE 1 AD P P P A A A 7THDay SAMPLE 2 AD P P P A A A 15 ± 5° C SAMPLE 3 AD P A A A A A SAMPLE 4 AD P P P A A A

[0094]

[0095] SAMPLE 5 AD P A A A A A Table 2: 7thDay Observation

[0096] SPECIMEN TYPE Eziprep LBC

[0097] SITE OF SPECIMEN Ecto & Endo Cervix

[0098] VDNA / LBINU003

[0099] REPORT NO Dated 16 / 3 / 2021

[0100] Day Sample

[0101] RTGP3

[0102] SAMPLE 1 AD P P P A A A 14TH Day SAMPLE 2 AD P P P A A A 30 ± 5° C SAMPLE 3 AD P A A A A A SAMPLE 4 AD P P P A A A

[0103]

[0104] SAMPLE 5 AD P A A A A A RFGP3

[0105] SAMPLE 1 AD P P P A A A 14TH Day SAMPLE 2 AD P P P A A A 15 ± 5° C SAMPLE 3 AD P A A A A A SAMPLE 4 AD P P P A A A

[0106]

[0107] SAMPLE 5 AD P A A A A A Table 3: 14thDay Observation

[0108] SPECIMEN TYPE Eziprep LBC

[0109] SITE OF SPECIMEN Ecto & Endo Cervix

[0110] VDNA / LBINU004

[0111] REPORT NO Dated 23 / 3 / 2021

[0112] Day Sample

[0113] RTGP4

[0114] SAMPLE 1 Ad AequacyD P P P A A A 21st Day SAMPLE 2 A AdequacyD P P P A A A 30 ± 5° C SAMPLE 3 AD P A A A A A C Cll Teompr

[0115] SAMPLE 4 AD C Cll Teompr Z Pone P P A A A SAMPLE 5 AD P Zone A A A A A RFGP4 C Rlleace.

[0116] C Rlleace

[0117] SAMPLE 1 AD P Ch.

[0118] Panges

[0119] Changes P A A A 21st Day SAMPLE 2 AD P P P A A A 15 ± 5° C SAMPLE 3 AD P A f O Inecrg. A A A A f O Inecrg.

[0120] SAMPLE 4 AD P P P A A A

[0121]

[0122] SAMPLE 5 AD P A A A A A S Ablqnorma.

[0123] Table 4: 21stDay Observation S Ablqnorma.

[0124] Gldblan anorma Gldan bl anorma SPECIMEN TYPE Eziprep LBC Ohlterma Ohltlerma neopasm SITE OF SPECIMEN Ecto & Endo Cervix l neopasm VDNA / LBINU05

[0125] REPORT NO Dated 30 / 3 / 2021

[0126] Day Sample

[0127] RTGP5

[0128] SAMPLE 1 AD P P P A A A 28TH Day SAMPLE 2 AD P P P A A A 30 ± 5° C SAMPLE 3 AD P A A A A A SAMPLE 4 AD P P P A A A SAMPLE 5 AD P A A A A A RFGP5

[0129] SAMPLE 1 AD P P P A A A 28TH Day SAMPLE 2 AD P P P A A A 15 ± 5° C SAMPLE 3 AD P A A A A A SAMPLE 4 AD P P P A A A

[0130]

[0131] SAMPLE 5 AD P A A A A A Table 5: 28thDay Observation SPECIMEN TYPE Eziprep LBC

[0132] SITE OF SPECIMEN Ecto & Endo Cervix

[0133] VDNA / LBINU006

[0134] REPORT NO Dated 06 / 4 / 2021

[0135] Day Sample

[0136] RTGP6

[0137] SAMPLE 1 LOW A A A A A A 35TH Day SAMPLE 2 AD P P P A A A 30 ± 5° C SAMPLE 3 LOW A A A A A A SAMPLE 4 AD P P P A A A SAMPLE 5 LOW A A A A A A RFGP6

[0138] SAMPLE 1 AD P P P A A A 35TH Day SAMPLE 2 AD P P P A A A 15 ± 5° C SAMPLE 3 A AdDequacy P A A A A A SAMPLE 4 AD P P P A A A

[0139]

[0140] SAMPLE 5 AD C Cll Teompr P A A A A A Table 6: 35thDay Observation Zone

[0141] Conclusion: The study confirms that the liquid-based cytology preservative composition of the present invention effectively maintains the integrity of cervical cytology samples. The f O Inecrg.

[0142] composition ensures sample stability for at least four weeks when stored at room temperature (30±5°C) and for more than five weeks when stored under refrigerated conditions (15±5°C), demonstrating its reliability for clinical use.

[0143] Gldan

[0144] bl anorma

[0145] 2. Accelerated Ageing Study of the Preservative Composition

[0146] Ohlter ma l neopasm Objective: The objective of this study was to evaluate the shelflife and functional stability of the liquid-based cytology preservative composition through accelerated ageing, in compliance with ASTM F1980-21: Standard Guide for Accelerated Ageing of Sterile Barrier Systems for Medical Devices. The study was designed to simulate two years of real-time storage stability within an expedited 91-day test period under controlled elevated temperature conditions.

[0147] Study Design: Accelerated ageing was performed by storing preservative vials at 55 ± 2 °C, corresponding to a real-time storage temperature of 25 °C. A conservative accelerated ageing factor (Q10= 2) was applied. Based on this, the accelerated ageing time (AAT) to simulate two years (730 days) of shelf life was calculated as 91 days. The test intervals were fixed at Day 0, Day 46, and Day 91. At each interval, samples were subjected to sterility testing in accordance with ISO 11737-2 and USP <71> guidelines to verify microbial integrity. Also, chemical stability testing was performed in accordance with IS 3025 standards. Sample and Methodology

[0148] • Sample description: Preservative vials from three manufacturing batches.

[0149] • Batch size: 15 vials per batch, with 5 vials tested at each interval.

[0150] • Test performed: Sterility testing (bacteria and fungi) using the membrane filtration method.

[0151] • Controls: Positive and negative controls included in all test runs.

[0152] • Procedure: Preservative vials were filtered through sterile 0.45 μm membranes, rinsed, and inoculated into Fluid Thioglycollate Medium (FTM) and Soybean Casein Digest Medium (SCDM). Incubation was performed at 30-35 °C (FTM) and 20-25 °C (SCDM). Daily observations were recorded during the incubation period.

[0153] Results

[0154] Sterility Testing

[0155] • Day 0 (Baseline): All samples across batches passed sterility testing. No microbial growth observed.

[0156] • Day 46: All samples maintained sterility. Positive controls showed expected microbial growth; negative controls remained sterile.

[0157] • Day 91: All samples continued to pass sterility testing, confirming microbial safety and integrity of the preservative medium at the accelerated end-point equivalent to two years of real-time storage.

[0158] Chemical Testing:

[0159] Chemical characteristics remained within acceptable ranges over the 91-day accelerated ageing period:

[0160] • pH: 6.17 - 6.49 (initial values 6.17-6.42).

[0161] • Water content: remained stable, with values showing consistent retention of aqueous fraction.

[0162] • Specific gravity: ~0.956 – 0.981 across all batches.

[0163] • Colour: remained clear and colourless throughout. Non-volatile residue: stable, with minor variation (0.010 - 0.040).

[0164] No deviations from the study plan occurred.

[0165] Conclusion: The accelerated ageing study confirmed that the liquid-based cytology preservative composition maintains sterility and chemical stability equivalent to a real-time shelf life of at least two years at 25 °C. Both microbiological and chemical integrity were preserved, confirming the robustness and reliability of the formulation under long-term storage conditions.

[0166] The present invention offers a formalin-free solution, eliminating the carcinogenic risks associated with conventional preservatives. Formalin, a known carcinogen, poses significant health hazards, including respiratory and skin irritation and long-term cancer risks. By removing formalin, the invention ensures a safer working environment for laboratory personnel and patients.

[0167] Additionally, the composition is free of polyethylene glycol (PEG), which in conventional solutions often emulsifies surface receptors, compromising immunocytochemistry results. By preserving surface receptor integrity, the invention ensures reliable and accurate receptorbased analyses, such as immunocytochemistry and other diagnostic techniques.

[0168] The invention also enhances molecular stability by preventing DNA and RNA degradation. This is a critical improvement over formalin-based preservatives, which often cause gene fragmentation, making them unsuitable for advanced molecular studies like PCR, HPV DNA analysis, and gene expression profiling. The invention ensures intact genetic material for reliable molecular analyses.

[0169] Furthermore, the multipurpose preservative is universally applicable across diverse sample types, including gynecological, non-gynecological, fine needle aspiration cytology (FNAC), and oral cytology specimens. This versatility simplifies laboratory workflows by reducing the need for multiple preservatives, thereby increasing operational efficiency and reducing costs. Also, the composition effectively preserves cellular morphology and structural integrity, ensuring artifact-free cytological smear preparations and clear staining results. This significantly enhances the diagnostic quality of preserved samples, providing improved outcomes for both routine and advanced cytological and molecular testing methodologies. It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the disclosure and are not intended to be restrictive thereof While specific language has been used to describe the disclosure, any limitations arising on account of the same are not intended.

[0170] The figures and the foregoing description give examples of embodiments. Those skilled in the art will appreciate that one or more of the described elements may well be combined into a single functional element. Alternatively, certain elements may be split into multiple functional elements. Elements from one embodiment may be added to another embodiment. For example, the order of processes described herein may be changed and are not limited to the manner described herein. Moreover, the actions of any flow diagram need not be implemented in the order shown; nor do all the acts need to be necessarily performed. Also, those acts that are not dependent on other acts may be performed in parallel with the other acts. The scope of embodiments is by no means limited by these specific examples.

Claims

WE CLAIM:

1. A liquid-based cytology preservative composition free of formalin, comprising:25% to 40% w / v of ethanol, acting as a cell preservative and antifungal agent; 0.25% w / v of Polysorbate-80, a non-ionic detergent stabilizing surface receptors and inactivating viruses;0.1M w / v of citric acid, enhancing DNA and RNA preservation and reducing degradation;0.2% w / v of 2-Chloroacetamide, providing antimicrobial activity;0.45% w / v of sodium chloride, maintaining cellular integrity;0.5% w / v of sodium hydroxide, maintaining pH balance; anddeionized water to make up the composition to a final concentration of 100% w / v.

2. The liquid-based cytology preservative composition as claimed in claim 1, wherein the pH of the solution is maintained at 6.4 ± 0.4, ensuring optimal stability and preservation of cellular morphology and molecular integrity.

3. The liquid-based cytology preservative composition as claimed in claim 1, wherein the composition is formalin-free and PEG-free, thereby preventing cytological staining interference, cellular hardening, and gene fragmentation.

4. The liquid-based cytology preservative composition as claimed in claim 1, wherein the composition is compatible with multiple sample types comprising at least one of Gynecological cytology samples, Non-gynecological cytology samples, fine needle aspiration cytology (FNAC) samples and oral cytology samples.

5. The liquid-based cytology preservative composition as claimed in claim 1, wherein the composition preserves surface receptors without disruption, enabling advanced testing methodologies such as immunocytochemistry, HPV DNA analysis, mRNA studies, and gene expression analysis.

6. The liquid-based cytology preservative composition as claimed in claim 1, wherein the ethanol concentration is optimized at 30% w / v for enhanced preservation and antifungal activity.

7. A method of preparing a liquid-based cytology preservative composition free of formalin, comprising:mixing 25% to 40% w / v ethanol with deionized water;adding 0.25% w / v Polysorbate-80 to the ethanol solution and stirring to dissolve; adding 0.1M w / v citric acid and ensuring complete dissolution; incorporating 0.2% w / v 2 -Chloroacetamide as an antimicrobial agent; dissolving 0.45% w / v sodium chloride and 0.5% w / v sodium hydroxide to maintain cellular integrity and pH balance;adjusting the final volume with deionized water to achieve 100% w / v concentration; andensuring the pH of the composition is maintained at 6.4 ± 0.4.

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