Title of Invention

A PROCESS FOR MANUFACTURE OF TWIN-WINDOW SLIDES HAVING LOW FLUORSCENCE AND A TWIN-WINDOW SLIDE MADE THEREBY USEFUL FOR COMET ASSAY

Abstract

A process for the manufacture of twin-window slides having low fluorescence useful for comet assay, characterised by subjecting a pre-cleaned sheet of slide glass such as herein described, having two covered windows of size in the range of 16 mm X 16 mm to 18 mm X 18 mm, to sand blasting with 50 to 75 micron sand, for a period of 15 to 25 minutes, at a pressure in the range of 35 to 45 psi, followed by cleaning, washing and drying by known methods.

Full Text

The present invention relates to a process for the manufacture of twin-window slides having low fluorescence and a twin-window slide made thereby useful for comet assay. The twin-window slides of the present invention having low fluorescence will find specific use for comet assay and also for applications in various other fields. The single cell gel electrophoresis (SCGE) or comet assay is a simple, sensitive, rapid and non-invasive technique, which permits quantitative and qualitative detection of the DNA damage in single cells. The simplicity of this assay, makes it a valuable tool to evaluate the DNA damage in virtually any type of eukaryotic cell and to assess the different kinds of DNA damage including double and single strand breaks, base damage, DNA cross-links, DNA adducts, DNA-DNA cross-links, DNA-Protein crosslinks, alkali-labile sites, apoptosis and also DNA repair processes. This technique has found wide acceptance in epidemiological and bio-monitoring studies to determine the DNA damage resulting from lifestyle, occupational and environmental exposures. The comet assay has found increasing use in the areas such as aging, human bio-monitoring, clinical epidemiology, oncology. However, an extremely useful application of the comet assay is in the field of genetic toxicology, to evaluate the in vitro and/or in vivo genotoxicity of chemicals. The extent of DNA damage occurring in any tissue e.g. blood, brain, bone marrow, liver or tumor tissue as well as cell type can be assessed simultaneously using comet assay. Besides humans and rodents, the comet assay has been extended to a variety of other biological species e.g. fish, molluscs, bullheads, algae and earthworm for eco-genotoxic effects. It has also been used to study DNA damage in plants cells and bacteria. Currently, comet assay is being increasingly used to study DNA damage in peripheral blood leukocytes, fibroblasts, alveolar macrophages, bladder cells, cervical epithelial cells, gastric mucosa and nasal cells, hepatocytes and germ cells of human or animal origin. In vitro genetic toxicology studies have also been conducted in human lymphocytes, mouse lymphoma cells, rat bone marrow and germ cells and also employing standard cell lines such as Chinese Hamster Ovary (CHO), V79, HeLa. In comparison to conventional cytogenetic techniques, comet assay is a rapid, simple and reliable method for screening of genotoxic potential of chemicals. Briefly, the single suspension of the cells of interest are embedded in low melting agarose and layered on to agarose pre-coated slides. Lysis of the cells, under high salt concentration, is then carried out to liberate the damaged DNA. The liberated DNA is subjected to unwinding under alkaline conditions, to allow DNA supercoils to relax and express DNA single strand breaks and alkali labile sites. This is followed by electrophoresis in highly alkaline (pH>13) conditions, which allows the broken ends to migrate under the effect of electric field, towards the anode. After neutralization, staining is done using fluorescent DNA dyes such as acridine orange, DAPI, propidium iodide, Hoechst 33258, SYBR green-1, YoYo dye etc. However, the most commonly used and stable dye is ethidium bromide. The slides are then kept in a humidified slide chamber till they are scored. The key component of this assay, however, is the glass slide. Currently, conventional glass slides (75mm X 25mm) are being used which have 19mm frosted end for labeling. Samples from each individual / test dose are processed in duplicate. A few of the drawbacks of using the hitherto known conventional slides are: 1. There is background fluorescence which interferes with the fluorescence of small DNA fragments which move out of the nucleus after electrophoresis in the Comet assay. Thus the results of the experiment get skewed especially when the extent of DNA damage is less. 2. There is inter and intra-slide variability among the duplicate slides for a sample. 3. The differences arising from the position of the duplicate slide in the electrophoresis apparatus are difficult to control. 4. Large amounts of samples are required for processing. 5. Large volumes (190ul per sample) of expensive (low melting point Agarose, Agarose, etc.) and hazardous chemical (e.g. ethidium bromide - 150ul per sample) and test compounds are used. 6. Big cover glasses (60mm X 24mm), which are very expensive, fragile and difficult to handle, are used. 7. In a single electrophoresis chamber (20cmX25cm) only 12 samples can be processed in duplicate thereby increasing the time for processing larger number of samples. 8. Archiving these slides also requires more space. Reference may be made to US patent no. 5,683,786 and 5,919,553, both titled: "Microscope slide having bar code indicia inscribed thereon". These patents describe a microscope slide having a glass plate, a marking surface coating on an area of the glass plate and indicia formed by etching away portions of the marking surface coating. The marking surface coating may comprise an epoxy resin material, a ceramic material, or any other substance capable of being affixed as a layer onto a glass surface. The indicia may include machine-readable indicia, human-readable indicia, or both machine-readable and human-readable indicia. In order to produce accurate, high-quality indicia, a laser directed by a computer controller may be used to etch away the portions of the marking surface coating. As per the hitherto known prior art, sand blasting / frosting is done to demarcate one end of the slide or for bar coding, for labeling and archival purposes. These single window slides are then used mainly for purposes such as histopathology, immunohistochemistry, immunofluorescence studies. However, no documentary evidence is available on the conditions of sand blasting / frosting. The type of sand, temperature and pressure play important roles in deciding the requisite quality of the blasting / frosting. Thus it is evident from the hitherto known prior art, that there is a definite need to provide a process for the manufacture of twin-window slides having low fluorescence and twin-window slides manufactured thereby. The main object of the present invention is to provide a process for the manufacture of twin-window slides having low fluorescence useful for comet assay, which obviates the drawbacks as detailed above. Another object of the present invention is to provide a slide with low background fluorescence for better and sensitive detection of DNA damage. Yet another object of the present invention is to provide a twin-window slide made by the process of the present invention, useful for comet assay, which obviates the drawbacks as detailed above. Still another object of the present invention is to provide an improved efficiency and cost effective slide, which will have two clear windows of size such as 18mmX18mm on a 75mm X 25mm roughened slides. Still yet another object of the present invention is, that the slide has the capacity to hold two samples and will minimize inter and intra-slide variability as identical conditions for duplicate slides will be maintained. A further object of the present invention is to eliminate the differences arising from the position in the electrophoresis apparatus. A still further object of the present invention is to reduce the quantity of samples and reagents required for processing, such as small volumes (80ul per sample) of expensive low melting point agarose, agarose, hazardous chemicals such as ethidium bromide - (60ul per sample) and test compounds are required. Another object of the present invention is to improve the efficiency as in a single electrophoresis chamber (20cmX25cm) 24 samples can be processed in duplicate thereby decreasing the time for processing larger number of samples. Still another object of the present invention is that archiving these slides require half space. In the present invention there is provided a process for the manufacture of twin-window slides having low fluorescence and a twin-window slide made thereby useful for comet assay. The twin-window slides of the present invention are improved and cost effective slides with twin clear windows for its application in comet assay, apoptosis, histopathology studies and the like. The twin clear windows are produced by sand blasting a pre-cleaned sheet of glass for 15 to 25 minutes with 50 to 75 micron sand at a pressure in the range of 35 to 45 psi. These slides have the added advantage of holding two samples on a single slide reducing the differences arising from positioning in the electrophoresis apparatus as well as inter and intra slide variability in the comet assay. Small amounts of expensive low melting agarose and hazardous chemicals are used. These slides are easy to handle, useful, cost effective and the results are reproducible. Larger number of samples can be processed at one time and they can be easily archived. Accordingly the present invention provides a process for the manufacture of twin-window slides having low fluorescence useful for comet assay, characterised by subjecting a pre-cleaned sheet of slide glass such as herein described, having two covered windows of size in the range of 16 mm X 16 mm to 18 mm X 18 mm, to sand blasting with 50 to 75 micron sand, for a period of 15 to 25 minutes, at a pressure in the range of 35 to 45 psi, followed by cleaning, washing and drying by known methods. In an embodiment of the present invention, the pre-cleaned sheet of slide glass is of size in the range of 25 mm X 75 mm to 25.4 mm X 76 mm and of thickness in the range of 0.96 mm to 1.2 mm. In another embodiment of the present invention, the pre-cleaning of the glass is done by thorough washing followed by heating at a temperature of the order of 100° C for a period of the order of 25 minutes. In yet another embodiment of the present invention, the sand blasting is done by sieved coast sand of mesh size 50 to 75 micron. In still another embodiment of the present invention, the two covered windows have two pieces of asbestos sheet to enable create the two clear windows on the slide post sand blasting. In still yet another embodiment of the present invention, the level of the sand blasted surface and clear windows are in the same plane and the pores are deep enough to hold agarose gel in place. In a further embodiment of the present invention, the number of clear windows is selected to be one or more, preferably two. Accordingly the present invention provides a twin-window slide useful for comet assay, manufactured by the process of the present invention as herein above described. In the process of the present invention a slide glass is cut into pieces of desired size with grinding of all edges. These slides are washed properly and heated at a temperature of the order of 100° C for about 25 minutes. Two pieces of asbestos sheet of desired window size are used during sand blasting to create the two clear windows. The sand blasting is done by sieved coast sand, which was sieved with fine mesh, with the help of strainer of size in the range of 50 to 75 micron. The blasting was done for 16 slides in a batch at a pressure in the range of 35 to 45 psi, for a period of15 to 25 minutes followed by known cleaning, washing and drying. To minimize the background fluorescence, precaution was taken with respect to the grain size of sand and the blasting pressure. Although, the level of the sand blasted surface and clear window were in the same plane, the pores were deep enough to hold the agarose gel in place. The present invention is illustrated in figures 1 and 2 of the drawings accompanying this specification. In figure 1 of the drawings is shown: (A) a conventional slide and (B) the twin window slide of the present invention. Figure 2 represents a flow chart showing the process of the present invention for manufacturing twin window slides with low background fluorescence. An illustrative experiment using conventional and twin-window slides of the present invention is described below to illustrate the unique points of the invention: Briefly, the cells (CHO, Human lymphocytes etc.) are treated in vitro and then washed before running the electrophoresis. Blood from control and exposed human / animal is collected in a heparinised tube and processed for isolation of lymphocytes. Other tissues are also processed for isolating and making a single cell suspension. To run the experiment the following materials are required: Disodium EDTA, Ethidium Bromide, Phosphate Buffered Saline (PBS) (Ca++, Mg++ free), Sodium Chloride (NaCI), Sodium Hydroxide (NaOH), Triton X-100, Tris Buffer, Normal Melting Agarose (NMA), Low Melting Point Agarose (LMPA), Coplin jars (opaque), Microcentrifuge Tubes, Micropipettor and Tips, Microscope Slides (75mm X25mm, with 19 mm frosted end for labeling), Coverslips (No. 1, 24 x 60 mm), Microscope Slide Tray (aluminum), Frozen Ice Packs, and Electrophoresis apparatus. The working solutions used are as follows: 1. Phosphate Buffered Saline PBS (Ca+2, Mg+2 free). 2. Lysing Solution (pH 10.0) consisting of NaCI, EDTA and Tris buffer. The final lysing solution is prepared fresh by adding 1% Triton X-100 and 10% DMSO to stock lysing solution. 3. Electrophoresis Buffer consisting of NaOH and EDTA. 4. Tris (pH7.5) is used as neutralization buffer. 5. Staining Solution: Ethidium Bromide (EtBr). The protocol for the experiment is as follows: (A). Preparation of base slides: 1. 1.0% Normal Melting Agarose (in Milli Q water) was prepared using a microwave. 2. The slides were dipped in methanol and burnt over a blue flame to remove the machine oil and dust. 3. While NMA is hot, the burnt slides were dipped up to one-third of the frosted area. The underside of the slide was wiped to remove agarose and the slide laid in a tray or on a flat surface to dry. The slides may be air-dried or oven dried at 50°C for quicker drying. 4. Slides were marked with lead pencil or diamond pencil before storage. 5. The slides were stored at room temperature until needed; avoiding humid conditions. (B). Preparation of comet slides: 1. 1% LMPA and 0.5% LMPA were prepared in PBS. This was microwaved or heated until near boiling when the agarose dissolves. 2. 50µl of cell suspension was mixed with 50µl PBS. Now 100µl 1% LMPA (37°C) was mixed well with the cell suspension and 80µl was loaded on to each base slide. A cover-slip was placed on it. The slides were kept on ice packs until the agarose layer hardens (~5 to 10 min). 3. Gently the cover-slip was removed and a third agarose layer (90 µl 0.5% LMPA) was added on to the slide. Cove-rslip was replaced and the slide kept on ice packs until the agarose layer hardens (~5 to 10 minutes). 4. Cover-slip was removed and the slide slowly lowered into, freshly made cold Lysing solution. It was protected from light and refrigerated for a minimum of 2-hours. For conducting the electrophoresis, fresh 1X electrophoresis buffer from the stock solutions was prepared before each run. Added 30 ml NaOH and 5 ml EDTA, to make up the volume to 2 litres and mixed well. The buffer reservoir was filled with freshly made Electrophoresis Buffer (pH>13). Electrophoresis of the slides under alkaline (pH>13) conditions was done as per the procedure described below: 1. Gently, slides were removed from the Lysing solution and placed side by side on the horizontal gel box near one end, sliding them gently as close together as possible. 2. Adjust the buffer level to completely cover the slides (avoid bubbles over the agarose). 3. The slides were allowed to sit in the alkaline buffer for 20 minutes to allow for unwinding of the DNA and the expression of alkali-labile damage. (The longer the exposure to alkali, the greater the expression of alkali-labile damage). 4. Turn on the power supply to 24 volts (~0.7-1.0 V/cm) and adjust the current to 300 milli amperes by raising or lowering the buffer level. Electrophoresis was carried out for 30 minutes. The optimal electrophoresis duration differs for different cell types. Different gel boxes require different voltage settings to correct for the distance between the anode and the cathode. A lower voltage, amperage and a longer electrophoresis time may result in increased sensitivity. 5. Turn off the power. Gently the slides were lifted from the buffer and placed on a drain tray. Drop wise coat the slides with Neutralizing Buffer (pH 7.4), and leave for at least 5 minutes. Drain the slides and repeat this procedure twice. 6. Slides were stained with 75µl 1X Ethidium Bromide (20µg/ml), left for 5 min and then dipped in chilled distilled water to remove excess stain. Coverslips were then placed over the slides and they are scored immediately or dried before staining as in step 7. 7. Drain the slides and keep them for 20 min in cold 100% ethanol or cold 100% methanol for dehydration. Air dry the slides and place them in an oven at 50°C for 30 min. Store dry. 8. When convenient, re-hydrate the slides with chilled distilled water for 30 min and stain with EtBr as in step 6 and cover with a fresh coverslip. Before viewing slides, blot away excess liquid on the back and edges. After scoring, remove coverslip, rinse in 100% alcohol to remove stain, let dry, and store for archival purposes if needed. The precautions taken are as follows: 1. The electrophoresis buffer should be chilled to prevent overheating during electrophoresis. 2. Voltage should be kept constant. 3. Neutralizing buffer should be added drop wise to prevent breakage of gel, and additional damage to DNA. 4. AH the above steps should be performed under dim yellow light to prevent DNA damage. The novelty of the present invention resides in providing a process for the manufacture of twin-window slides having low fluorescence useful for comet assay, which obviates the drawbacks of the hitherto known glass slides used for such purposes. The novel features include providing a slide with low background fluorescence for better and sensitive detection of DNA damage. Twin-window slides useful for comet assay, which are cost effective and more efficient. Slides having the capacity to hold two samples, thus minimizing inter and intra-slide variability as identical conditions for duplicate slides will be maintained and eliminates the differences arising from the position in the electrophoresis apparatus. Reduces the quantity of samples and reagents required for processing. Helps improve the efficiency by increasing the number of samples that can be run in a single electrophoresis chamber (20cmX25cm) from 12 samples (75mmX25mm slides in duplicate) to 24 samples (75mmX25mm Twin Window Slides). The novel features of the present invention have been realized by the non-obvious inventive steps which comprise subjecting a pre-cleaned sheet of slide glass having two covered windows to sand blasting with 50 to 75 micron sand, for a period of15 to 25 minutes, at a pressure in the range of 35 to 45 psi. The following examples are given by way of illustration of the working of the invention in actual practice and therefore should not be construed to limit the scope of the present invention. Example -1 For making the desired twin-window slides as per the process of the present invention a sheet of glass of 1.2 mm thickness having the following composition was taken: SiO2 72.80 % Al2O3 1.40% Fe2O3 0.08 % CaO 6.60 % MgO 4.20 % R2O 14.62% SO3 0.30 % and alkalis, Na2 O & K2 O The above said glass sheet of 1.2 mm thickness was cut into pieces of 75 mm x 25 mm with grinding of all edges. These slides were washed properly and heated at 100° C for 25 minutes. Two pieces of asbestos sheet of size18mm X 18mm were used during sand blasting to create the two windows. The sand blasting was done by coast sand, which was sieved with fine mesh, with the help of strainer. The blasting was done at a pressure of 40 psi (2.72 atmospheric pressure) for approximately 20 minutes for 16 slides in a batch. To minimize the background fluorescence, precaution was taken with respect to the grain size of sand and the blasting pressure. Although, the level of the sand blasted surface and clear window were in the same plane, the pores were deep enough to hold the agarose gel in place. Example -2 The twin window slides can be used for assessment of DNA damage and repair using the comet assay. The types of damage to DNA that can be studied are DNA-DNA cross links, DNA-protein cross links, Alkali labile sites, DNA single and double strand breaks. The slides were dipped in methanol and burnt over a blue flame to remove the machine oil and dust. Then base slides were prepared by dipping the slides up to one-third of the frosted area in hot (60 degree C) 1.0% Normal Melting Agarose (dissolved in Milli Q water). The underside of the slide was wiped to remove the agarose and the slide placed in a tray or on a flat surface to dry. (The slides were stored at room temperature until needed; avoiding humid conditions.) A single cell suspension of the cells of interest was obtained {in vivo) or after treatment {in vitro) in the medium suitable for the cells or phosphate buffered saline (PBS). 50ul suspension is diluted 1:1 with 50ul of 1% low melting agarose. 40ul each is loaded onto each window (18mmX18mm) of the base slide and a cover slip (22mm X 22mm) placed on it. The slide was kept on ice for 5 minutes to allow the gel to solidify. Then, the cover-slip was removed and 50ul of 0.5% low melting agarose (LMPA) at 37 degree C was added onto the slide and again covered with a cover-slip. The slide was again put in ice to allow the gel to solidify. The cover slips were removed and the slides put into chilled lysing solution. The lysing was done for at least two hours and then the slides were drained and put into an electrophoretic chamber containing freshly prepared chilled electrophoresis buffer (pH>13). Unwinding of DNA was done for a minimum of 2 hours and then electrophoresis was carried out for 30 minutes at 0.7 V/cm and 300 mA current. Slides were then neutralized with chilled neutralizing solution thrice each for 5 minutes. The slides were then dipped in chilled distilled water to remove excess alkali and stained with ethidium bromide for 5 minutes. The slides were again dipped in chilled distilled water to remove excess stain, and a fresh cover-slip (22mm X 22mm) was put on the windows. The slides were read within 24 hours on a fluorescent microscope. Example -3 Assessment of apoptosis using comet twin window slides: The comet TW slides can also be used effectively to study apoptosis in individual cells. The slides were pre-coated with 50ul of 0.6% agarose by spreading it evenly with a pipette tip on the windows. These were dried and stored. 10ul of suspension of cells of interest were mixed with 60ul of 0.6% agarose and layered on to the clear windows of the pre-coated slides. The cover-slip was put and the slide was put on ice or at room temperature to allow solidification of gel. A third layer of 80ul SFR agarose (2%) was then added and again allowed to solidify. The cover-slips were removed and the slides put into lysing solution for 10 minutes at room temperature. Neutralizing was done twice each 30 minutes. These slides were then dried in an oven at 50 degree C. Later on these were stained with 30ul of YOYO-1 dye and covered with a cover glass. Slides were scored on a fluorescent microscope using blue filter at a magnification of 200X and 400X. 500 cells were counted from each window. Example - 4 A typical experiment to assess the DNA damage in human lymphocytes by the alkaline comet assay was performed using the conventional and the twin window slides. The cells were treated with different concentrations of a known mutagen, ethyl methane sulphonate. Our data demonstrates that the DNA damage observed in the cells on conventional as well as the twin window slides was almost the same. The results are depicted in figures 3, 4 and 5 of the drawings accompanying this specification. Figure 3 shows the comparison of Olive Tail Moment values between conventional slide and twin window slide of the present invention. Figure 4 shows the comparison of Tail DNA values between conventional slide and twin window slide of the present invention. Figure 5 shows the comparison of Tail length values between conventional slide and twin window slide of the present invention. The main advantages of the present invention are: 1. Provides a process for the manufacture of twin-window slides having low fluorescence useful for comet assay. 2. Provides a slide with low background fluorescence for better and sensitive detection of DNA damage. 3. Provides twin-window slides useful for comet assay, made by the process of the present invention, which are cost effective and more efficient. 4. Provides an improved efficiency and cost effective slide which will have two clear windows of 18mmX18mm on a 75mm X 25mm roughened slides. 5. Provides slides having the capacity to hold two samples, thus minimizing inter and intra-slide variability as identical conditions for duplicate slides will be maintained. 6. Eliminates the differences arising from the position in the electrophoresis apparatus. 7. Reduces the quantity of samples and reagents required for processing, such as small volumes (80ul per sample) of expensive low melting point agarose, agarose, hazardous chemicals such as ethidium bromide - (60ul per sample) and test compounds are required. 8. Improves the efficiency as in a single electrophoresis chamber (20cmX25cm) 24 samples can be processed in duplicate thereby decreasing the time for processing larger number of samples. 9. Archiving these slides require half space. 10. Reduces per sample cost to approximately one fourth of the current cost as detailed below table 1. Table 1 (Table Removed) We claim: 1. A process for the manufacture of twin-window slides having low fluorescence useful for comet assay, apoptosis, and histopathology studies, which comprises providing a pre- cleaned sheet of glass slide of size in the range of 25 mm X 75 mm to 25.4 mm X 76 mm and thickness in the range of 0.96 mm to 1.2 mm, having two covered windows with asbestos sheet of size in the range of 16 mm X 16 mm to 18 mm X 18 mm, wherein the composition of the glass comprising; Si02 72.80 % Al203 1.40% Fe2 03 0.08 % CaO 6.60 % MgO 4.20 % R20 14.62% S03 0.30 % and alkalis, Na2 O & K2 O, subjecting the said glass slide to sand blasting with 50 to 75 micron sand, for a period of 15 to 25 minutes, at a pressure in the range of 35 to 45 psi, followed by cleaning, washing and drying by known methods. 2. A process as claimed in claim 1, wherein the pre-cleaning of the glass is done by thorough washing followed by heating at a temperature of the order of 100 °C for a period of the order of 25 minutes. 3. A process as claimed in claim 1, wherein the level of the sand blasted surface and clear windows are in the same plane and the pores are deep enough to hold agarose gel in place.

Documents:

1870-DEL-2004-Abstract-(24-03-2011).pdf

1870-del-2004-abstract.pdf

1870-del-2004-Claims (02-09-2011).pdf

1870-DEL-2004-Claims-(24-03-2011).pdf

1870-del-2004-claims.pdf

1870-DEL-2004-Correspondence-Others-(24-03-2011).pdf

1870-del-2004-correspondence-others.pdf

1870-DEL-2004-Description (Complete)-(24-03-2011).pdf

1870-del-2004-description (complete).pdf

1870-DEL-2004-Form-1-(24-03-2011).pdf

1870-del-2004-form-1.pdf

1870-del-2004-form-2.pdf

1870-DEL-2004-Form-3-(24-03-2011).pdf

1870-del-2004-form-3.pdf

1870-del-2004-form-5.pdf

1870-del-2004-Pre Grant Opponent Doc..pdf

1870-DEL-2004-Pre-Grant Opposition-(02-09-2011).pdf

1870-DEL-2004-Pre-Grant Opposition-(05-08-2011).pdf