Diagnosis of Fanconi anemia (congenital myelodysplasia)--dioxobutane (dibutyl epoxide) detection assay
Diagnosis of Fanconi anemia (congenital myelodysplasia)--dioxobutane (dibutyl epoxide) detection assay
Fanconi anemia (FA) is an autosomal recessive syndrome characterized by progressive bone marrow failure and an increased risk of malignancies, particularly acute myelogenous leukemia and squamous epithelial cell carcinoma.
Operation method
Diagnostic test for Fanconi anemia dioxybutane after birth
Materials and Instruments
Peripheral blood Move 1. Place a drop of peripheral blood (18-25 drops; 0.4-0.5 ml) from a 10 ml syringe with an 18 1/2 G needle into a 25 cm2 tissue culture flask containing 10 ml of RPMI/15% FBS complete medium. Two portions of the medium were configured for DEB study and two portions were used as untreated controls (4 portions in total). The medium was incubated for 24 h (Appendix 31). 2. Dispense PBS into three 15 ml centrifuge tubes: tube 1, 10 ml; tube 2, 6 ml; and tube 3, 4.5 ml. 3. Before adding medium, add 10 ul of common DEB to tube 1, cap, and mix well. Transfer 4 ml from tube 1 to tube 2, cap and mix. Transfer 0.5 ml (500ul) from tube 2 to tube 3, cap and mix well. 4. Take 25 u1 of solution from each of tube 3 and add it to two peripheral blood cultures that have been incubated for 24 h (from step 1; the final concentration in the culture flask is 0.l ug/ml.). Incubate for another 48-72 h. 5. Add 2 ml of colchicine 1ul/ml to every 10ml of medium and incubate for 20 min. 6. Transfer the liquid from each culture flask to a 15 ml centrifuge tube with a conical bottom, cover and centrifuge at 150 g for 10 min. 7. Remove most of the supernatant, flick the tube with your finger to resuspend the flaky precipitate in the remaining supernatant, and carefully add 5 ml of pre-warmed 0.075mol/L Ka to swell the cells but do not break them. Incubate for 10 min in a water bath at 37°C. Centrifuge for 10 min at 150 g at room temperature. 8. Remove the supernatant and gently add 1 ml of fresh fixative without washing up the sediment. Remove the fixative and repeat the step. 9. Immediately flick the wall of the tube several times with your index finger to break up the flaky precipitate. Quickly add fixative to keep the cells in suspension and prevent them from clumping. The final volume is 8-10 ml. Use a Pasteur pipette to break up the clumps. Allow to stand at room temperature for 30 min. 10. Centrifuge at 150 g for lOmin at room temperature, remove supernatant and leave 3-5 ml to suspend the flaky precipitate. Allow to stand at room temperature for lOmin. This step can be repeated once or twice immediately, or after allowing the cells in the fixative to stand overnight at room temperature. 11. Centrifuge at 150 g for lOmin at room temperature. remove the supernatant, break up the precipitate, and add an appropriate amount of fixative (0.25~0.5 ml) to reach a desired concentration for use in the preparation. An ideal cell concentration will result in the vast majority of cells in one microscopic field of view with no crowded mid-stretch. 12. From an appropriate height, drop a few drops of cell suspension onto a clean slide so that the mid-division phase is well dispersed (Unit 4.1). A minimum of 6 slides should be made from one bottle of medium (it is recommended that non-diagnostic cells be used for this exercise). This step is the key to success. 13. Observe the slide under an inverted microscope to determine the quality and quantity of the intermediate division phase. Stain the slide with Giemsa (Support Option 2). 14. Analyze 50 to 100 giemsa-stained midphases per DEB-treated specimen (25 midphases for highly broken cases; Appendix 3K). Chromosomes were counted and the number and type of chromosomes with structural abnormalities were recorded. If broken chromosomes exceed the normal range above, analyze the untreated specimen. Photograph the abnormal cells after the analysis is complete. 15. Interpret the results according to the published results as a basis and in the case of DEB-induced chromosome breaks in ectopic peripheral blood lymphocytes under PHA stimulation. Table 8.7.1 shows baseline and treatment culture data obtained from normal and affected individuals DEB (1,3-diepoxybutane) is a potential carcinogen and some precautions must be taken when handling this compound. This section describes how to use and properly dispose of the waste. Workspace. Any work with DEB must be done in a chemical fume hood or a Level n Biology Safe Work Closet. Wear lab coats and gloves when working with DEB. Keep a bottle of 6mol/L HCl (50 ml) on hand at all times to inactivate DEB if it is accidentally poured. Cell culture. Cell culture operations involving DEB should be done in a level n biological safety cabinet. Once the cells have been immobilized, the operation can be performed outside the safety cabinet. Dispose of discarded media and wash solutions with 6mol/LHCl and treat as hazardous biological waste. Previously used pipettes and culture flasks should be rinsed with 6mol/L HCl and placed in a high-pressure hazardous bio-bag. Micropipette tips should also be disposed of in vials containing 6mol/L HCl. DEB stock solutions should be disposed of as hazardous chemical waste and not diluted or chemically inactivated. Used plastic utensils should be rinsed with 6md/LHCl and disposed of in a high-pressure hazardous bio-bag. Culture media and cleaning solutions should also be treated with 6mol/LHCl and disposed of as hazardous biological waste. Giemsa Stain is a histologic stain that has a special affinity for nuclei and chromosomes. It is simple to make and causes unbanded nuclei and chromosomes to turn a slight red to purple color. 1. Dissolve one tablet of Gurrs buffer pH 6.8 in 1L of water. 2. Add 4 ml of fresh Giemsa's Stain to a vat containing 46 ml of Gunrs' Buffer. The solution can be used for 1h or to stain 20 slides before a new solution has to be prepared. 3. Stain the intermediate chromosome slides for 5 min. and rinse several times in a vat of Gurrs buffer (step 1). 4. Rinse several times in a vat of water. Dry in air and check the intensity of staining of the slide. The staining intensity should be sufficient and the cracks and broken areas on the chromosomes are not easily stained (non-staining). 5. Load coverslips onto slides using Permount Tissue Sealer. If peripheral blood is not available, the test can be performed by skin tissue biopsy fibroblast culture. The test can also be performed by taking a few small pieces of lung tissue or skin from an aborted or stillborn child. Because higher concentrations are too toxic to FA fibroblasts, it is difficult to harvest enough mid-division phases for study. 1. 0.5 ml of living tissue in DMEM complete medium/20% FBS solution is placed on a 60 mm tissue culture dish and cut into pieces, 1 mm in size, with a sterile scalpel. 2. Take at least three other 60 mm tissue culture dishes and use the tip of a sterile scalpel to mark several horizontal and vertical lines on the bottom of the dish. Place a few drops of DMEM complete medium/20% FBS solution onto the biopsies. Place 5 or 6 slices of living tissue on each 60 mm tissue culture dish using a sterile Pasteur pipette. Place the tissue at the intersection of the horizontal and vertical lines. 3. Incubate the petri dishes without adding any other tissue culture medium for 15-30 min to allow the tissue pieces to adhere to the dish. Add 5 ml of DMEM complete medium/20% FBS solution to each Petri dish and incubate the medium. 4. Replace the medium every 3~4d, but do not disturb the tissue slices. Observe the growth of the cells in the medium using an inverted microscope (it will take 1 week to 1 month to observe the better growing cells). 5. When large fibroblasts can be seen around the tissue sheet in the Petri dish, treat the cells with 1X insulin/EDTA/15% FBS and pass them on (Appendix 31), taking care not to remove the living tissues when they are able to continue to grow in the medium. The cells were transferred to 25 cm2 sterile tissue culture flasks containing DMEM complete medium/15% FBS. 6. When the first generation of cells is full grown, treat again with trypsin (Appendix slide). The second generation culture is divided 1:3 into DMEM complete medium/15% FBS solution. At least two second generation cultures are full of cells when performing DEB tests for FA patients. 7. Inoculate cells in 25 cm2 flasks with 5 ml of DMEM complete medium/15% FBS solution to a density of 3X105 cells per flask (two treated and untreated control flasks are prepared for this test). Incubate for 24 h. 8. Dilute the DEB as previously described (basic protocol, steps 2-3) and add a fourth tube, dispensing 4.5 ml of PBS. Fibroblast cultures will be added with a final step of dilution, i.e., 0.5 ml (500ul) of DEB from tube 3 is added to tube 4. 9. Remove 254 from tube 4 and add to 10 ml of fresh DMEM complete medium/15% FBS solution. 10. Incubate the cells until they are nearly full grown (approximately 72 h). 11. Trypsin digest the cells (Appendix 31) and pass them on 1:2 or 1:3 to fresh complete DMEM/15% FBS medium. This medium is DEB-free and is used to collect sufficient mitotic cells for cytogenetic studies. Restriction medium, which contains fresh, diluted DEB, is reintroduced. 12. Collect cultured cells at the first mitotic epoch (24-48 h of passaging; the cells are near mid-mitotic) and add 1 ml of colchicine at a concentration of 1ug/ml to the DEB-treated cells 3 h prior to collection, while using the non-DEB-treated cells as a control (in 5 ml of medium). 13. Digest the cells with trypsin, transfer the liquid from the culture flask to a 15 ml sterile centrifuge tube, cover and centrifuge at 150 g for 10 min. 14. Remove most of the supernatant, resuspend the precipitate in the remaining supernatant and slowly add 5 ml of 0.051mol/L KCl. Incubate for lOmin in a water bath at 37°C. Centrifuge for lOmin. 15. Fix the cells on prepared slides as described previously (basic protocol, steps 9-12; in step 9, slowly add 3-5 ml of fixative). 16. 100 untreated and DEB-treated Giemsa-stained mid-division phase cells were examined for broken chromosomes as described previously (basic protocol, step 13, supporting protocol 2). Interpret the results according to the assessment established for baseline and DEB-induced chromosome breaks in FA patient fibroblasts. FA can be diagnosed at term, but with some risk, by culturing chorionic villus sampling (CVS) to obtain trophoblast cells at 9 to 12 weeks of gestation or amniocentesis to obtain amniotic fluid cells at 15 to 17 weeks of gestation FA can also be diagnosed prenatally with fetal blood, as previously done with peripheral blood cultures. 1. Configure fetal cell culture medium (Module 8.1 and Module 8.2) in 25 cm2 culture flasks. 2. When there are several large, fast-growing clones in the culture flask, do not overgrow the clones, digest the cells with trypsin, and perform the first passaging culture at 1:2 or 1:3 (Appendix 31). Incubate for 24 h. 3. Replace the growth medium (from two different first passages) with fresh growth medium with 0.01 ug/ml DEB in at least two culture flasks. If possible, replace the first-passage medium with medium containing 0.1 ug/ml DEB in two additional culture flasks, with the same dilution as before (Option 丨, steps 8~9). The fluid in the remaining culture flask serves as an untreated control for the baseline chromosome breakage study. 4. Incubate the medium, harvest and fix the cells, and prepare the interim chromosome smears as previously described (Option 1, starting at step 10). 5. A total of 100 mid-stage schizonts are examined in each group (baseline group, DEB-treated group; 50 mid-stage schizonts from each of the two first-passage culture flasks in each group). Interpretation of the results was based on assessments established for baseline and DEB-induced chromosome breaks against FA fetal cells obtained by CVS or amniocentesis. For more product details, please visit Aladdin Scientific website.
RPMI 15% FBS Complete Medium Dioxobutane Colchicine KCl Fixative
Syringes Tissue culture flasks Tubes Centrifuge Centrifuge Inverted microscope
