Expert consensus on the quality control of in situ culture technique for amniotic fluid cells on slides (2025 Edition)

1. In order to reduce the time interval between amniocentesis and prenatal diagnosis of Pompe's disease microchemical techniques were used for assay of acid alpha-1,4-glucosidase activities in cultured amniotic fluid cells. 2. Microtechniques used on homogenates of cultured amniotic fluid cells enabled the waiting period to be reduced to 2-3 weeks. 3. When dissected lyophilized groups of 200-300 cultured cells were analyzed, a prenatal diagnosis was possible at about 10 days after amniocentesis. 4. The acid alpha-1,4-glucosidase activity in the amniotic fluid supernatant is not informative in prenatal diagnosis of Pompe's disease. 5. Conditions of cell cultivation such as length of time in culture were found to influence markedly the acid alpha-1,4-glucosidase activity in cultured amniotic fluid cells. 6. For a reliable prenatal diagnosis of metabolic disorders primary cultures of control amniotic fluid cells should be used and the analytical results from the pregnancy at risk should be compared with primary cultures of control amniotic fluid cells and with those in cultured fibroblasts from heterozygous carriers, and an affected sibling from the particular family.

Objective: To explore the potential of flow cytometry in the prenatal exclusion or confirmation of Fanconi anemia (FA). Methods: Indications for prenatal diagnosis were (1) FA-negative family history, but suspicious ultrasound findings such as radial ray aplasia, (2) FA-positive family history, but without knowledge of the affected gene and/or mutation. Amniotic fluid (AF) cell cultures and umbilical cord (UC) blood cultures were assayed for typical cell cycle changes (G2-phase accumulations) without and with mitomycin C (MMC) treatments using single- and dual-parameter (BrdU-Hoechst) flow cytometry. Results: Single-parameter flow cytometry correctly identified 2 positive and 9 negative cases on the basis of MMC sensitivity of cultivated AF cells. Likewise, 8 negative and 2 positive cases were correctly predicted using bivariate flow cytometry of 72-hour UC blood cultures. In contrast, bivariate flow cytometry applied to AF cells grown in the presence of bromodeoxyuridine (BrdU) yielded false-positive and false-negative results.Conclusions: Single-parameter flow cytometry of AF cell cultures and bivariate flow cytometry of UC cell cultures have the potential to correctly predict the affected status in cases at risk for FA, whereas bivariate flow cytometry proved unreliable when applied to BrdU-substituted AF cell cultures. Cases with a low a priori risk (e.g. sonographic finding of radial ray abnormalities and negative family history) would benefit most from flow cytometry as a rapid and economical prenatal screening procedure.

Objective To evaluate amniotic fluid cell inheritance and re-culture in cases of amniotic fluid with abnormal traits. Methods From January 2014 to December 2018, 15 cases of amniotic fluid with abnormal traits in the First Affiliated Hospital of Anhui Medical University were selected in amniocentesis.Amniotic fluid cells were routinely seeded and cultured for 10 days, then subcultured into other culture bottles.The number of cells and karyotyping after harvesting were counted. Results Seven of 15 cases of amniotic fluid color were slightly darker, 3 cases were pink as water washed meat, and 5 cases were light brown or brown.The average number of cells in original bottles was (2.40±5.87)×105/mL, the average number of cells in inheritance bottles was (2.76±0.64)×106/mL.All 15 samples in the cell inheritance bottles got satisfactory results in cell karyotype analysis. Conclusion Amniotic fluid cell inheritance and re-culture can increase the number of cells in amniotic fluid cell culture and improve the success rate of karyotyping. Key words: Amniocentesis; Amniotic fluid; Prenatal diagnosis; Cell culture techniques; Chromosomes; Cytogenetic analysis; Karyotyping

We describe a simple synchronization culture technique of amniotic fluid (AF) cells to yield many earlier mitotic divisions with extended chromosomes. AF cell samples obtained by amniocentesis were cultured in the usual manner. Thirty hours after the first subculture, they were exposed to excess thymidine (0.5 mM). This cell cycle block was released by adding deoxycytidine (10 microM) 18 hr after synchronization. At exactly 7.5 hr after the release, the cultures were treated with Colcemid (0.02 microgram/ml) for 20 min then harvested. The mitotic index and the ratio of cells in the earlier mitotic stages were much higher in the synchronized cultures than in the control cultures. The same favorable effects were obtained also in AF cell cultures by combining this technique with ethidium bromide or actinomycin D treatment. The technique was less toxic to the cells, and was simple and reproducible. It was successfully applied to prenatal cytogenetic diagnosis of 2 families with a subtle inherited chromosome abnormality, so it is recommended for high-resolution banding analysis of AF cells and possibly chorionic villus samples.

Prenatal diagnosis of many inherited diseases is based on studies using cultured AF cells, and therefore morphologic studies on amniotic fluid (AF) cells have increased in importance. However, the nature, morphology and origin of AF cells which grow in tissue culture remains unsettled. In this study AF cells from the first trimester of pregnancy were examined by light, transmission (TEM) and scanning electron microscopy (SEM).Amniotic fluids (15-20 weeks of gestation) were examined from 11 patients undergoing therapeutic saline abortions and from four intact amniotic sacs with a foetus. Percentages of dead cells were determined using Trypan blue stain, and differential cell counts were made using cytocentrifuge sedimented AF cells stained by Wright's stain and PAP stain.

Detection of Duchenne muscular dystrophy gene products in amniotic fluid and chorionic villus sampling cells

To assess the value of combined chromosomal karyotyping and chromosomal microarray analysis (CMA) and/or copy number variation sequencing (CNV-seq) for the prenatal diagnosis for women with advanced maternal ages, and to explore the challenges of prenatal genetic counseling brought by the types of fetal CNVs and uncertainty of related phenotypes. A retrospective analysis was carried out on 1 841 women with advanced maternal age who underwent interventional prenatal diagnosis at the Prenatal Diagnosis Center of Xiamen University Affiliated Women and Children's Hospital from January 2017 to December 2020. Routine chromosomal karyotyping analysis and CMA/CNV-seq detection were carried out. CMA/CNV-seq had detected pathogenic variants in 2 cases which had failed karyotyping analysis. Two hundred and twenty one fetal chromosomal abnormalities were detected by karyotyping analysis, among which 187 were detected by CMA/CNV-seq. CMA/CNV-seq analysis of 23 cases with balanced chromosome structural aberrations and 10 cases with low proportion mosaicisms (including a marker chromosome) had yielded a negative result. In addition, 26 cases (26/1 841, 1.4%) with pathogenic CNVs were discovered among those with a normal karyotype, of which 13 (50.0%) were recurrent CNVs associated with neurocognitive impairment, with 22q11.21 microdeletions and microduplications being the most common types (26.92%). The combination of karyotyping analysis and CMA/CNV-seq not only increased the rate of prenatal diagnosis, but also complemented with each other, which has facilitated genetic counseling and formulation of prenatal diagnosis strategy for the affected families.

Extract: We have developed a randomized, multivariable block design for quantitative evaluation of conditions for amniotic fluid (AF) cell attachment to culture surface and subsequent growth rate. We report the use of this method to determine optimal fetal calf serum (FCS) concentration in the culture medium. The AF cell growth, in the form of discrete colonies, was scored by counting the number of colonies and measuring their diameters. A two-way analysis of variance of the preliminary experiment data (5–30% PCS) demonstrated a significant difference among AF cells grown at different percentages of FCS (P < 0.0005). From a studentized range analysis we concluded that 5% FCS is inferior to higher percentages. No difference was demonstrated among 15-30% FCS. Based on these data, a supplementary experiment was planned to compare 10 and 15% FCS. The 15% FCS yielded at least 0.31 colonies/ml AF more than did 10% FCS. We conclude that 15% FCS is sufficient for AF cell culture using our technique. Close agreement of the estimates of standard deviation of the preliminary and supplementary experiments (7.38 and 7.25) indicates that the assumptions required for our experimental design are correct and that our technical procedures are highly reproducible. A larger series of 76 AF specimens was used to correlate patient data with AF cell growth. We have shown that AF cell growth is independent of: (1) gestational age between 11 and 24 weeks; (2) maternal age between 14 and 42 years; (3) maternal ethnic group. A mean of 6 colonies/ml AF was obtained. Speculation: Development of highly efficient and reliable procedures for culturing AF cells depends upon statistically valid, quantitative assessment of all variables surrounding amniocentesis and subsequent cell culture. Using a randomized, multivariable block design, optimal culture procedures and conditions can be established and standardized which result in increased culture success rate and decreased time requirement for prenatal detection.

Variation in lysosomal enzyme activity during growth in culture of human fibroblasts and amniotic fluid cells

Sixteen pregnancies at risk for Gaucher disease -- six with the Norrbottnian form, one with a juvenile form with a similar clinical course to the patients from Norrbotten and nine with the infantile from -- have been monitored by the assay of beta-glucosidase activity in cultivated amniotic fluid cells with natural labelled glycosylceramide as substrate. Two methods of cultivation were compared in respect of their effect on the activity of lysosomal enzymes. No significant difference was found between the two marker enzymes, beta-galactosidase and N-acetyl-beta-glucosaminidase, but the beta-glucosidase activity was significantly higher in the cells cultivated with one of the methods. In four of the pregnancies at risk, the beta-glucosidase activity in the cultivated amniotic fluid cells was less than 5% of that in the two control materials. These fetuses were regarded as affected with Gaucher disease and were aborted. Differentiation between controls and Gaucher heterozygotes was not possible in cultivated amniotic fluid cells. The diagnosis of Gaucher disease in the amniotic fluid cells was confirmed in three of the four cases by the assay of the beta-glucosidase activity in the liver nd brain of the aborted fetuses. The glucosylceramide content of the liver from two aborted fetuses was not augmented. The beta-glucosidase activity was examined in seven placentas from pregnancies at risk for Gaucher disease and found to be in agreement with that in the cultivated amniotic fluid cells.

The cell morphology of long-term cultures of amniotic fluid cells from 10 fetuses with a neural tube defect (NTD) and three with omphalocele was examined and compared to 30 long-term cultures of normal amniotic fluids as well as a long-term culture of human fetal brain. Cultures from the amniotic fluids of the fetuses with NTD and omphalocele showed cells with the same general characteristics as normal amniotic fluid cells. However, the cultures of amniotic fluid cells from NTD pregnancies had an additional cell type also seen in fetal brain culture. This was a neuroblast-like cell, with small rounded refractile morphology and long branching processes forming clusters of varying sizes which lay on top of large flat cells. These neuroblast-like cells diminished in number with time in culture and were not present in subcultures. Their possible neuronal origin is discussed.

The growth-promoting activities of three different bovine sera have been compared in primary and secondary amniotic fluid cell cultures. In secondary amniotic fluid cell microcultures, aseptically collected calf serum (CS) was slightly, though not significantly more effective than fetal calf serum (FCS) in promoting DNA synthesis, while newborn calf serum (NCS) was significantly less effective than either CS or FCS. All three sera were optimally effective at concentrations of 10 per cent (v/v). Significant variation in quality occurred within four batches of each of CS and FCS, but not within four batches of NCS. A selected batch of CS was significantly more effective in promoting the growth of primary amniotic fluid cell cultures than were a number of batches of FCS then in routine laboratory use. It is suggested that CS may serve as an effective and economical alternative to FCS in the culture of amniotic fluid cells, thereby expanding the scope of serum batch testing. A possible explanation for the varying growth-promoting activities of different sera is discussed.

Amniotic fluid cells have been widely used in prenatal diagnosis; however, there is great heterogeneity of the cells and their origin. In this study we analyze the karyotype and release of human chorionic gonadotropin (hCG), human chorionic somatomammotropin (hCS), free estriol (E 3), prolactin (PRL) and progesterone (P) of amniotic fluid cells from primary cultures of six normal and two anencephalic fetuses. In all the amniotic fluid samples there was release of hCG; in one amniotic fluid, in which several tetraploid colonies were found. PRL and P were also released. The heterogeneity of amniotic fluid cell morphology and their hormone release in culture was confirmed. The presence of hormones like hCG supports the trophoblastic origin of some amniotic fluid cells from normal and anencephalic fetuses. Other hormones, such as PRL and P could be used in the differential diagnosis between the karyotype of fetal membranes and the true fetal karyotype. Amniotic fluid cell cultures used in prenatal diagnosis yielded second trimester placental cells without any elaborate methods that could be used as cell models for hormone studies.

Chromosomal karyotyping analysis has been considered as the gold standard for cytogenetic diagnosis and an effective measure for preventing birth defects. However, conventional karyotyping analysis relies heavily on manual recognition, which is time-consuming and labor-intensive. The application of an efficient intelligent chromosomal karyotyping precise auxiliary diagnosis system in clinical practice can significantly reduce the analysis time and greatly improve the efficiency, increase the detection rate for low-percentage mosaicisms, and promote homogenization between laboratories. This can effectively enhance the capacity and level of cytogenetic diagnosis. With the continuous application of such system in the field of karyotyping analysis, a substantial amount of clinical application data and experience has been accumulated. This consensus has been formulated after multiple rounds of discussion by experts from the clinical application collaboration group of the efficient intelligent chromosomal karyotyping precise auxiliary diagnosis system. It aims to provide a reference for peers to better utilize intelligent auxiliary diagnosis systems during chromosomal karyotyping analysis and promote the standardized development of karyotyping analysis technology.

To assess the application value in prenatal diagnosis using karyotype analysis combined with BACs-on-Beads (BoBs) assay. Nine hundred sixty five pregnant women were subjected to amniocentesis, chromosomal karyotype analysis and detection of BoBs were employed simultaneously for abnormal number of chromosomes and 9 chromosome microdeletion syndrome in prenatal diagnosis. Fifty cases common chromosome aneupoidies were successfully detected by both karyotype analysis and BoBs which included 31 cases of trisomy 21,10 cases of trisomy 18 and 9 cases with sex chromosome abnormality. BoBs in addition detected 1 case of DiGeorge-1 microdeletion syndrome and 1 case of 7q11.23 microduplication syndrome. All 9 fetuses with chromosome abnormalities detected by karyotyping were missed by BoBs, including 2 cases of marker chromosomes,4 cases of chromosomal translocation,1 case of chromosomal inversion, 2 cases of Sex chromosome mosaicism; 2 cases of fetal inherited from the parents,7 cases for novel mutations. Karyotype analysis combined with BoBs dedtection is a rapid, effective and highly accurate prenatal diagnosis model that may should be widely used in clinical diagnosis.