The effect of amniotic fluid cell morphology
We found that spindle-shaped amniocytes (Fig 1 A–C) can be transfected by SV40LT, and that round-, elliptical-, and polygonal-shaped amniocytes (Fig 1 D–H) cannot be immortalized using SV40LT (Fig 1).
G418 selection of positive clones
A total of 24 positive clones were selected from groups A, B, and C using 50–400 mg/L G418. These cells were mostly spindle-shaped or germinative during early growth (Fig 2A). Positive cells were obtained from all groups after selection for 7–15 days, (Fig 2B). Clones consisting of single-layer, adherent spindle cells were obtained after growth in high glucose DMEM containing 10% FBS (Fig 2C). Of these clones, 22 showed logarithmic growth for 10–15 generations. The chromosome karyotypes of the clones included six cases of trisomy 21, four cases of trisomy 18, three cases of 45,X, two cases of 47,XXY, and one case of each of trisomy 13, 48,XXYY, 46, XY, t(1; 20)(q32; p21), 46, XX, t(6; 7)(p25;q11.2), 46, XX,t(3; 8)(q25; p23), 46,XY,t (15;17) (q11;q11), and 47, XX, +mar.
Comparison of SV40LT transfection efficiency in Groups A, B, and C
Eleven, three, and ten positive clones were obtained in groups A, B, and C, corresponding to transfection rates of 18.3 (11/60), 5.0 (3/60), and 16.7% (10/60), respectively. Chi-square analysis revealed that the difference between groups A and B was significant (χ2 = 3.96 > χ2 (1, 0.05) = 3.84), but the difference between groups A and C was not (χ2 = 0.58 < χ2 (1, 0.05)) (Table 1).
Table 1. SV40LT amniocyte transfection in groups A, B, and C
Groups
|
Transfection method
|
No. of
amniocytes
|
No. of
positive clones
|
Positive rate (%)
|
A
|
mediated by trypsin
|
60
|
11
|
18.3
|
B
|
mediated by PT67 cell culture medium
|
60
|
3
|
5.0
|
C
|
mediated by liposomes
|
60
|
10
|
16.7
|
Total
|
|
180
|
24
|
13.3
|
Karyotype analysis and aneuploidy detection in amniocyte lines
Three different amniocyte lines were randomly selected at generations 10–15 for chromosome preparation, karyotype analysis, and FISH. The karyotypes of the transfected amniocyte lines were identical to those of their primary cells: 46, XY, t(1; 20)(q32; p21) (Fig 3A), 46, XX,t(3; 8)(q25; p23) (Fig 3B), and 47,XX,+mar (Fig 3C). Mutations occurred in chromosomes 7, 18, 20, and 22 in 3% of the amniocyte lines generated from one sample, 45,XY,-14,der(7),der(9),der(18),der(20),der(22) (Fig D). Subsequently, 10th to 15th generation amniocyte lines comprising 47,XY,+13 and 47,XXY were assessed using FISH and the 13/21 dual-color and 18/X/Y tri-color probes. The fluorescence signals of chromosomes 13, 18, 21, X, and Y in these amniocyte lines were identical to those in their corresponding primary cells (Fig 4 A–D).
Application of chimera quality-control cells in FISH
The chimeric quality-control cell line with 47,XY,+13, 47,XY,+18, 47,XY,+21, and 48,XXYY theoretically present in equal proportions was tested by three different technicians for five consecutive days using the same conditions as those used for the clinical samples. The results (n = 5, mean ± 1 SD) are shown in Table 2 and Figure 5. The proportion of 47,XY,+18 and 48,XXYY cells observed statistically differed from the predicted 25% as measured by technicians A (P < 0.05) and C (P < 0.01), respectively.
Table 2. Application of chimera quality control cell lines in FISH (n = 5, mean ± 1 SD)
Karyotype
|
Theoretical ratio (%)
|
Actual ratio (%)
|
A
|
B
|
C
|
Average△
|
47,XY,+13
|
25
|
24.8±1.9
|
25.2±2.2
|
25.4±1.3
|
25.1±0.3
|
47,XY,+18
|
25
|
28.6±1.1*
|
25.2±1.3
|
24.8±0.8
|
26.2±2.1
|
47,XY,+21
|
25
|
25.2±1.3
|
25.6±1.1
|
26.4±0.5
|
25.7±0.6
|
48,XXYY
|
25
|
25.4±1.5
|
25.6±1.1
|
21.8±0.8**
|
24.3±2.1
|
△: The mean and standard error of A, B, C
*: Compared with the theoretical ratio, P < 0.05; **: Compared with the theoretical ratio, P < 0.01.
Application of chimera quality-control cells in karyotype analysis
The chimera karyotype was composed of four different amniocytes with chromosome abnormalities. Theoretically, the chimera karyotype was 46, XY, t(1; 20)(q32; p21)[10] / 46, XX, t(6; 7)(p25;q11.2)[20] / 46, XX, t(3; 8)(q25; p23)[30] / 47, XX, +mar[40] and the predicted proportion of each chimera karyotype was 9, 18, 33, and 43%. The observed proportion of the four 46, XY, t(1; 20)(q32; p21), 46, XX, t(6; 7)(p25;q11.2), 46, XX, t(3; 8)(q25; p23), and 47, XX, +mar karyotypes in the chimera was 8, 23, 33, and 35%, respectively, after testing 20 subjects. The observed proportion of 47, XX, +mar was 8% lower than predicted. Additionally, 47, XX, + mar was misdiagnosed as 47, XX, +19 in one instance, providing a misdiagnosis rate of 5.0% (1/20) (Table 3).
Table 3. Chromosome karyotype quality analysis in 20 chimeric quality-control cell line subjects
|
Karyotype of chromosome in chimera
|
Subject number
|
Chimera distribution percentage (%)
|
Chimera prediction percentage (%) A
|
Chimera quality assessment mean (%) B
|
Difference value (A-B) (%)
|
Misdiagnosis mean (%)
|
Misdiagnosed karyotype number
|
46, XY, t (1; 20) (q32; p21)
|
20
|
10
|
9
|
8
|
1
|
|
|
46, XX, t (6; 7) (p25; q11)
|
20
|
20
|
18
|
23
|
-5
|
|
|
46, XX, t (3; 8) (q25; p23)
|
20
|
30
|
33
|
33
|
0
|
|
|
47, XX, + mar
|
20
|
40
|
43
|
35
|
8
|
5
|
[47, XX, + 19 (1)]
|