Amniocentesis cell culture chromosome examination
Due to harmful chemicals, X-ray exposure, environmental factors, advanced pregnancy, close relatives, etc., leading to diseases caused by the variation in the number, morphology, structure and combination of chromosomes during pregnancy. Chromosome examination of amniotic fluid cells has great specific significance for prenatal diagnosis of chromosomal diseases.Basic Information
Specialist Category: Maternal Examination Check Category: Genetic Testing (DNA)
Applicable gender: whether women are fasting: fastingAnalysis results:
Total number of chromosomes: 46. Autosome: 22 pairs (number 1-22). Sex chromosomes: XY for men and XX for women. Male karyotype: 46, XY. Female karyotype: 46, XX.
Results that are different from normal values are positive, suggesting a congenital malformation or other congenital disease.
The total number of chromosomes is 46.
22 pairs of autosomes (numbered 1 to 22).
The sex chromosome male is XY and the female is XX.
Male karyotype 46, XY.
Female karyotype 46, XX.Clinical significance
Abnormal chromosome number such as Down syndrome (trisomy 21), trisomy 18, trisomy, trisomy 38, trisomy 21, congenital testicular hypoplasia syndrome, XXY synthesis Sign, Turner syndrome, X trisomy syndrome and multiple X body syndrome.
Abnormal chromosome structure such as 4p partial monomer syndrome, 5p partial monomer syndrome, 9p partial trisomy syndrome, 9q partial monomer syndrome, 21q partial monomer syndrome, 22q partial monomer syndrome, 22q partial trisomy Syndrome, etc.Positive results may be diseases: congenital ovarian hypoplasia, pediatric cerebral palsy, pediatric Down's syndrome, pediatric cerebral palsy, pediatric pascal syndrome, Diggolger syndrome, pediatric meow syndrome, pediatric congenital testicular development Incomplete, genetic disease considerations
1. Chromosome examination should take amniotic fluid specimens at 16-20 weeks of gestation.
2, these congenital genetic diseases have increased, lacking, translocation, inversion and other quantitative and structural abnormalities, often manifested as a syndrome. Most cases, such as multiple malformations, growth retardation and mental retardation, lead to fetal abortion, premature birth or stillbirth.Inspection process
(1) Take 15 to 30 ml of amniotic fluid for 16 to 20 weeks of pregnancy, and centrifuge at 1000 r/ml for 10 min.
(2) Discard the excess supernatant, leave 1 ml of amniotic fluid and precipitated cells, and gently disperse into a cell suspension.
(3) Transfer into a 25 ml square culture flask, add 3 ml of a medium having a pH of 6.5 to 6.8 (containing penicillin 100 U/ml, streptomycin 100 μg/ml) and 1 ml of calf serum, and incubate in a 37 ° C incubator.
(4) A large number of fibroblast-like or epithelioid cell colonies were observed after 7-10 days of culture. The fresh medium can be exchanged at this time.
(5) When the cell colonies are expanded into pieces and there are many translucent circular dividing cells, colchicine can be added to make the final concentration 0.1-0.3 μg/ml, and then cultured in a 37 ° C incubator for 5-6 h. (The above procedures are all carried out under aseptic conditions). Harvesting standards: 1 with fibroblasts as the main type, the growth of the cells; 2 with 10x eyepieces and 20x objective observation, the growing cell clones cover 1 or more complete fields of view; 3 see more than 10 translucent Round cells and more than 10 double round bright dividing cells.
(6) If the cells are not growing vigorously, the growth cycle is not uniform or the cells are aging, and the harvesting standard is not reached, the subculture can be continued in the original bottle. Method: Under sterile conditions, the culture solution was first poured, and a few drops of 2.5 g/L sterile trypsin solution were added, and the mixture was shaken and poured. Further, 5 to 10 drops of trypsin were added, and gently shaken at 37 ° C for about 5 minutes to cause the adherent cells to fall off. 4 ml of fresh medium containing calf serum was added, and the culture was allowed to stand at 37 ° C for 4 to 5 hours. Carefully change the culture medium and continue the culture. It can be harvested 3 to 5 days after the passage.
(7) Transfer the culture solution to a graduated centrifuge tube, add 1 ml of ED-TA-trypsin solution to the flask, place in a 37 ° C incubator for 5 min, and then rinse the adherent cells with an elbow pipette (2.5 g/ can also be used) L trypsin solution digestion). The cells detached from the bottle wall were poured into a centrifuge tube, mixed with the original culture liquid phase, and the culture flask was washed with a small amount of warm physiological saline, and the washed cells were also poured into the centrifuge tube and centrifuged at 1000 r/min for 10 min.
(8) Aspirate the supernatant, add 3 to 5 ml of 0.075 mol/L KCl hypotonic solution pre-warmed at 37 ° C, and set at 37 ° C for 10 to 15 min.
(9) Pre-fixation, fixation and preparation are the same as the preparation method of peripheral blood cell chromosome specimens.Not suitable for the crowd
1. There were signs of abortion during pregnancy.
2. When the body temperature exceeds 37.5 °C.
3. Early exfoliation of the placenta, abdominal infection and purulent.Adverse reactions and risks
(1) Maternal injury: puncture needle stab wound blood vessels caused by abdominal wall hematoma uterine subserosal hematoma. Occasionally, amniotic fluid enters the maternal blood circulation from the puncture hole and causes amniotic fluid embolism. The bladder was not emptied before the puncture, and the bladder was injured.
(2) Injury of the fetus, placenta and umbilical cord: The puncture needle can damage the fetus and can cause bleeding, and the placenta and umbilical cord can also cause bleeding or hematoma. Therefore, the source of bleeding should be identified when taking hemorrhagic amniotic fluid. If you suspect that you are from a fetus, you should continue to listen to the fetal heart.
(3) Leakage of amniotic fluid: postoperative amniotic fluid leaks from the pinhole, causing too little amniotic fluid, affecting fetal development, and even causing miscarriage or premature birth.
(4) abortion or premature birth: the incidence of abortion or premature delivery is 0.1% -0.2%, often occurs within one week after surgery, even after the puncture, premature rupture of membranes leads to premature delivery.
(5) intrauterine infection: there may be maternal fever after surgery. Intrauterine infection can cause abnormal fetal development, or even fetal death. Therefore, amniocentesis should be strictly aseptic.