Peripheral blood lymphocyte culture and chromosome preparation technology have been widely used in cytogenetic research and chromosome disease diagnosis. Because this technology needs a long cell culture time and complicated preparation process, it is easily affected by factors such as temperature, humidity, PH value, hemolysis and coagulation, which leads to the failure of the experiment. Therefore, controlling each experimental step is very important for the success or failure of the experiment. However, some specimens can not be karyotyped, and the reasons for their failure are emphatically analyzed.

I. Experimental Steps and Control Measures

1. Specimen collection, inoculation and culture:

Routine disinfection was carried out on the blood collection site of the patient, and about 3mL of blood was collected with a 5mL disposable sterile syringe or blood collection needle, and 5mL sterile heparin lithium anticoagulation tube was injected, inverted and mixed evenly. Strengthen the training of specimen collectors in outpatient and inpatient departments. Specimens must be collected aseptically to avoid hemolysis and coagulation.

In the biosafety cabinet, extract anticoagulant with disposable sterile syringe (2.5mL) and inoculate it into culture bottles (including 5mL of human peripheral blood lymphocyte culture solution), each bottle is 0.4mL, and at least two bottles are inoculated. After inoculation, gently shake horizontally for several times and mix well. Stand upright in two incubators at 37 0.5℃ for 66 ~ 72 hours, and observe whether the culture solution has coagulation, hemolysis or bacterial growth the next day. All culture solutions and reagents used for cell culture and cytological treatment must be pre-tested, and a new batch of reagents must be verified between batches after arrival.

2. Cell harvesting and production:

Before the end of culture, add colchicine to the culture medium in the biosafety cabinet, with the final concentration of colchicine of 0. 1~0.5μg/mL, gently shake it evenly, and put it in an incubator at 37 0.5℃ for1.5h. After colchicine treatment, carefully take out the culture bottle from the incubator and suck the culture with a straw. Centrifuge: 1500r/m×65438+ Discard the supernatant, add 8 ~10 ml of 0.075mol/L KCl hypotonic solution incubated at 37℃, blow it into cell suspension with a straw, and put it in a water bath at 37℃ for 25 minutes. Add 1.5mL Carnot stationary liquid (methanol: acetic acid 3: 1) to each centrifuge tube, gently suck and blow, and let it stand at room temperature 10min. Centrifuge: 1500 rpm × 10 min, and discard the supernatant. Then, add 8mL of Carnot stationary solution into the centrifuge tube, immediately blow it into a single cell suspension with a straw, leave it at room temperature for 30min, centrifuge: 1500r/m× 10min, discard the supernatant, and repeat this for three times. Colchicine treatment time is too long, there are many splinter cell and short chromosomes; On the contrary, it is few and slender. These two situations are not suitable for observing morphology and counting, so it is necessary to accurately grasp the concentration and time of colchicine. Hypotonia makes erythrocyte membrane rupture and lymphocytes swell, so the concentration and time of hypotonic treatment should be appropriate. However, the mixed cells must be light after fixation, otherwise it will cause cell rupture and chromosome loss.

Drop 0.2~0.5 mL of fresh stationary liquid into the centrifuge tube, gently blow the lymphocyte mass into cell suspension with a straw, take out borneol from the refrigerator, add 2~3 drops of suspension to each tablet, and bake at 75℃ for 2 ~ 3 hours. The slide must be cleaned in ice bath, otherwise the chromosome will not disperse well. In the production process, if it is found that the cells are not swollen, the cell membrane is not broken, the chromosomes are clustered and cannot be stretched, the fixed time can be extended for several hours or the proportion of glacial acetic acid can be appropriately increased.

3. Bundling and dyeing

Pour 50ml of115m phosphate buffer with pH value of 7.4 into a vertical dyeing vat, add 0.5ml of 2.5% trypsin solution and mix well. After pancreatin digestion, it was stained with 10% Giemsa dye solution for 3 minutes, then the glass slide was taken out with tweezers, and both sides were gently washed with tap water, and then dried with a hair dryer. Pretreatment should be done before each tape development to determine the correct tape development time, and all slides should not be treated blindly. To judge the banding effect, it is necessary to observe the split phase with moderate length, and the next banding time cannot be determined only by the banding effect of 1 and 2 split phases.

Second, the analysis of the reasons for the failure of training

As far as 20 15 is concerned, so far, there are 1500 specimens, and 10 specimens can not be used for chromosome analysis after culture and production. The preparation of peripheral blood chromosomes is a manual project, and the process is complex, and every link should be strictly controlled. The staff in this room have undergone strict training and can only take up their posts after passing the exam. The causes of these 10 unqualified samples have all been ruled out as operator errors. The facilities and environment of the laboratory meet the experimental requirements. So we made a telephone call back to these six patients and found that one common feature of * * * was the use of anti-inflammatory drugs, especially for pediatric inpatients.

Antibiotics may inhibit lymphocyte transformation and affect the quality of culture. For this part of patients, we suggest to draw blood again at least two weeks after stopping taking the drug. There are 4 cases of patients with increased lymphocyte count after blood collection and culture, which can be used for chromosome analysis. 1 The patient still had no effective mitotic phase after blood sampling again, and was told by phone that he was still taking anti-inflammatory drugs of traditional Chinese medicine. There are still 5 patients who have not been reviewed.

Three. Corrective and preventive action

For the failure of peripheral blood chromosome preparation, we first rule out the influence of human error and laboratory facilities, and then consider whether it is the cause of the case itself under the same conditions. Therefore, the corrective measures are: inform patients that the specimen culture is unqualified, and the reason may be related to the use of anti-inflammatory drugs. It is recommended to stop taking drugs for at least two weeks before taking blood.

At present, our laboratory always asks the patient's medical history before collecting samples, so we can prevent the culture failure rate by asking whether to use anti-inflammatory drugs. I told you that the chromosome examination is not urgent. You can check it after stopping taking the medicine.

In a word, in order to prepare chromosome specimens for karyotype analysis, every step of cell culture, production and banding can not be ignored. It is necessary to constantly sum up experience in the operation process in order to improve the success rate of peripheral blood chromosome specimen preparation.

Genetic group Yang Li/Wen

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