A Novel External Quality Assessment of Chromosomal Karyotype Analysis Based on Chimera Image Assembly.

OBJECTIVE: This study aimed to establish a new external quality assessment (EQA) of chromosomal karyotype analysis. METHODS: Chimeric assembly A1 was established by collecting chimeric chromosome images prepared artificially from chromosomally abnormal amniocytes remaining after prenatal diagnosis. Chimeric assembly B1 and nonchimeric assembly C1 were constructed through the collection of chimeric and nonchimeric chromosome images from prenatal diagnosis, respectively. Then, chromosome images were selected randomly from assemblies A1, B1, or C1 to send to 20 technicians via email to verify the validity of a new EQA of chromosomal karyotype analysis. RESULTS: According to the EQA of 20 technicians, 47,XX,+mar from assembly A was easily misdiagnosed as 47,XX,+19 or 47,XXY, and 45,XX,t(13;22) (q10;q10) was misdiagnosed as 45,XX,13S+,-22. The total misdiagnosis rate was 3.8%. For assembly B, 46,X,+mar and 46,X,idic(Y) were easily misdiagnosed as 46,XY and 46,X,+mar, respectively. In addition, some testers missed 47,XXX in 47,XXX[2]/46,XX[48], as well as 47,XX,+18 in 46,XX [47]/47,XX,+18[3], and 45,X and 47,XXX in 46,XX[47]/45,X[2]/47,XXX[1]. The total misdiagnosis rate was 4.2%. All karyo-types from assembly C were correctly diagnosed, although incorrect descriptions used for 4% of cases. CONCLUSION: The quality of chromosome karyotype analysis can be comprehensively evaluated by a new EQA based on assembly A1 or B1.

A Quality Assurance Scheme for Prenatal Detection of Aneuploidy Based on Developing Chimeric Quality-Control Cells.

BACKGROUND: The shortage of quality-control materials caused by non-renewable utilization of rare disease samples is the key factor to limit the quality control of prenatal molecular diagnosis. This study aimed to prepare aneuploid amniocyte lines for the development of quality control cells for fluorescence in situ hybridization (FISH)-mediated detection of aneuploidy. METHODS: Recombinant SV40LTag-pcDNA3.1(-) vectors were transfected into 47,XY,+18 amniotic fluid cells with the use of liposomes. After culturing, these cells were mixed with primary amniocytes with the karyotype 46,XY to prepare four groups of chimeric quality control cells comprising recombinant cells with the karyotypes 47,XY,+18 and primary cells with 46,XY, with theoretical ratios of 47,XY,+18 cells at 5%, 10%, 20%, and 40%. Subsequently, the chimeric quality control cells were tested as clinical samples by three technicians to examine their feasibility for use as internal quality controls (IQC) for FISH detection. RESULTS: After being immortalized by the SV40 large T antigen gene (SV40LT), these aneuploid amniocytes can be cultured indefinitely to prepare chimeric quality control cells. The actual ratio of the 47,XY,+18 cells was identified by FISH to be 1.5 ± 1.1%, 10.3 ± 1.0%, 19.9 ± 0.4%, and 40.8 ± 0.3%, respectively, and the fluorescence signals of chromosomes 13, 18, 21, X, and Y in these cells were consistent with that of the primary cells. CONCLUSIONS: The present study may resolve the shortage of quality control cells in the prenatal detection of chromosomal aneuploidy and may provide a foundation for IQC-based detection in FISH.

A novel use for Levey-Jennings charts in prenatal molecular diagnosis.

BACKGROUND: The goal of this study was to determine whether Levey-Jennings charts, which are widely used in clinical laboratories, can be used to create standardized internal quality controls (IQCs) for prenatal molecular diagnosis. METHODS: Aneuploid amniocyte lines with trisomy 13, 21, and 18, and 47,XXY were established by transfection with SV40LTag-pcDNA3.1(-)and combined at different ratios to generate aneuploidy chimeric quality-control cell mixtures A to H. These quality-control cells were then used to calculate the [Formula: see text], [Formula: see text] ±1 standard deviation (SD), [Formula: see text] ±2 SD, and [Formula: see text] ±3 SD values to develop standardized IQCs for methods used for the prenatal diagnosis of aneuploidies such as FISH. RESULTS: Methods for constructing aneuploid amniocyte lines were developed and a set of quality-control cells (A-H) were prepared. The [Formula: see text] ±1 SD, [Formula: see text] ±2 SD, and [Formula: see text] ±3 SD values of these quality-control cells for trisomy 13 and 21 were 10.2 ± 1.7, 10.2 ± 3.4, and 10.2 ± 5.1, and 90.3 ± 2.3, 90.3 ± 4.6, and 90.3 ± 6.9, respectively. Based on the values and Levey-Jennings charts, a set of standardized IQCs for prenatal diagnosis such as FISH were established. CONCLUSIONS: This method resolves the problems of a shortage of quality-control materials and a lack of quality-control charts in prenatal molecular diagnosis such as NIPT, NGS, aCGH/SNP, PCR, and FISH. Levey-Jennings chart-based IQCs for prenatal diagnosis such as FISH can be used to easily monitor whether IQC results are within acceptable limits, and then infer whether the diagnostic results for clinical samples are reliable. We expect that this standardized IQC will be useful for a wide range of molecular diagnostic laboratories.

Desirable quality-control materials for the establishment of qualified external quality assessment on prenatal diagnosis of chromosomal aneuploidies.

OBJECTIVE: To prepare desirable quality-control materials for the establishement of qualified external quality assessment on fluorescence in situ hybridization (FISH)-detected prenatal diagnosis of chromosomal aneuploidies. METHODS: Four types of amniotic fluid cell suspensions (13-trisomy, 18-trisomy, 21-trisomy and 47,XXY) were mixed together by ratio to produce mosaicism with the percentages of each aneuploidy as 10%, 20%, 30% and 40%, respectively. After being stored in liquid nitrogen of -196 °C for six months, randomly selected samples were incubated in 37 °C water, followed by cultivation, hypo-osmosis and fixation. Finally, FISH detetion was applied on them before and after external laboratory mailing, in step with detection on conventional case samples. RESULTS: Before mailing, the positive rates of each aneuploidy described above were 12.8%, 23.6%, 33.8%, 44.0%, while 12.6%, 23.8%, 34.0%, 43.5% after mailing. t-test, criteria for stability assessment of quality-control materials in CANS-GL03:2006, showed no significant effect of external mailing on mosaicism since corresponding t values are lower than threshold with significance level α as 0.05 and degree of freedom as 10. CONCLUSION: As FISH detection showed, the mosaic cell strains prepared in current study exhibited excellent stabilities after cryopreservation in -196 °C, subculture, hypo-osmosis, fixation and external laboratory mailing, demonstrating them as reliable and promising quality-control materials for the establishment of a qualified external quality assessment on prenatal diagnosis of chromosomal aneuploidies.

[Establishment of cell lines for quality control of prenatal genetic diagnosis by SV40LT gene transfection].

OBJECTIVE: To establish a cell lines for quality control of prenatal genetic diagnosis. METHODS: The recombined SV40LTag-pcDNA3.1(-) vector was constructed and transfected by lipidosome into human amniotic fluid cells with common aneuploidy. Positive clones were screened by G418, and the immortality of transfected cell line was identified. RESULTS: Cell line with karyotype of 46, XY, t(8;19)(q24.3;q13.1) from primary amniotic fluid cells was established. Karyotype analytical results indicated that the cell line at its 15th generation maintained the same karyotype of its primary cell. CONCLUSIONS: Gene SV40LT can lead to immortality of amniotic fluid cells, which contributes to preparing cell lines for internal and external quality control in prenatal genetic diagnosis.

An external quality assessment scheme for prenatal detection of rare chromosomal abnormalities.

OBJECTIVE: To improve the accuracy of prenatal cytogenetic diagnosis by establishing an external quality assessment (EQA) scheme for rare, or subtle, structural chromosomal abnormalities. METHOD: Typical metaphase images of rare chromosomal abnormalities along with an anonymized clinical history were sent to 35 prenatal diagnosis laboratories. The laboratories were required to provide independent reports using current nomenclature. Evaluation of reports was based on established criteria for quality assessment of karyotype analysis. RESULTS: Totally 6 kinds of typical rare chromosomal abnormalities were collected. 35 laboratories were enrolled with a response rate of 94.29%. The overall analytical accuracy is 82.20%. This is comparable with our former EQA scheme using established lymphocyte cell lines with rare abnormal chromosome karyotypes (χ²=0.065, P=0.799). CONCLUSION: Introduction of efficient EQA for prenatal genetic diagnosis is currently imperative, due to the limited performances of karyotype analysis of rare chromosomal abnormalities. Metaphase images could be used as a simple and effective material for EQA and to improve the quality of prenatal genetic diagnosis.