Materials and Methods
Retrospective, deidentified clinical data from pregnant women who underwent NIPT through Illumina Laboratory (formerly Verinata Health) from Feb. 15, 2012, through Nov. 15, 2012, at 6 US medical centers (3 of which have multiple clinic locations) was collected by trained study coordinators on a standardized case report form. Participating centers were early adopters of NIPT from various locations (California [n = 2], Nevada, Minnesota, Virginia, and Connecticut). Before the introduction of NIPT into practice, all centers used standard forms of first and/or second trimester prenatal screening (eg, first trimester combined, integrated or sequential screening) as recommended by the 2007 ACOG guideline. Trained genetic counselors and/or maternal-fetal medicine specialists presented NIPT as an additional screening option and provided pre- and posttest counseling to their patients. The study protocol was approved by Chesapeake Institutional Review Board and a waiver for collection of informed consent and authorization for use and release of deidentified clinical information from patient charts was granted. Three of 6 centers obtained an additional approval from their local institutional institutional review board.
Study-site coordinators reviewed relevant clinical records for specific data elements pertaining to the study. Completed case report forms were returned to the central clinical research team at Illumina for double data entry into a custom study database (Microsoft Access, Redmond, WA). The following data was collected: maternal age and gestational age at time of NIPT, pretest indications, prenatal screening results (biochemical screening with or without NT measurement), medical history relevant to the pregnancy (eg, maternal diabetes, hypertension, thrombophilia), NIPT test results, prenatal invasive procedure indications, and cytogenetic results, if performed, and pregnancy outcome if known. Finalized datasets were exported into a Microsoft Excel file for further analysis and generation of tables. Each site was also asked to collect overall rates of CVS and amniocentesis procedures for their practice for 1 year before the introduction of NIPT and for the year following its introduction. Data was provided by centers in 1 of 3 ways: billing statistics (n = 3), laboratory accessioning (n = 1), or an internal physician database (n = 2).
All tests had been processed at the College of American Pathologists–accredited, Clinical Laboratory Improvements Amendments–licensed, Illumina clinical laboratory in Redwood City, CA. NIPT by massively parallel sequencing of cfDNA was performed and results reported as described by Futch, et al. Samples received before July 2, 2012, were classified for aneuploidy status of chromosomes 21, 18, and 13. For chromosomes 21, 18, and 13, the sequencing laboratory classified the sample in 1 of 3 ways: aneuploidy detected, no aneuploidy detected, or unclassifiable (borderline value). On or after July 2, 2012, an additional testing option for Monosomy X (MX) became available for indication of cystic hygroma. For the MX option, the classification was either MX detected or MX not detected. Unclassified was not a result option for MX. Laboratory cancellations of tests were due to either administrative reasons or technical reasons. Providers were notified if a result was cancelled and offered the option to submit a second sample.
The 3 sites located in the west were grouped together as were the 2 sites in the east. Both groups were compared with each other and with the site located in the Midwest. Summary statistics were used for demographic variables. Data comparisons between the 3 groups were performed using the 2-proportion Z-test to calculate P values.
Results
Data forms for 1490 patients were returned of which 1477 comprised the analysis cohort for this study. Thirteen cases were excluded because they were marked twin gestation for which the test was not indicated during the time of testing. NIPT results were reported by the laboratory for 1455 (98.5%) patients, of which 51 (3.5%) showed aneuploidy detected for chromosome 21 (n = 26), 18 (n = 10), 13 (n = 9), or MX (n = 6). An unclassifiable result was reported in 41 (2.8%). There were 10 (0.7%) tests cancelled because of administrative reasons (insufficient quantity [n = 6], duplicate order [n = 1], patient or physician request [n = 1], compromised sample in transit [n = 1] and improper labeling [n = 1]). Twelve samples (0.8%) were cancelled because of technical reasons; excessive DNA content (n = 10) or insufficient cfDNA extraction (n = 2).
Patient demographics, indications for NIPT and invasive procedure use are shown in Table 1 . Of 1477 patients who underwent NIPT; 693 (47%) were from the west, 522 (35.3%) were from the east, and 262 (17.7%) were from the Midwest. The mean maternal age at the time of NIPT was greater in the west (36.6 years) compared with the east (33.8 years; P < .0001) and Midwest (33.5 years; P < .0001). Mean gestational age was lower in the west (14.1 weeks) compared with the east (16.1 weeks; P < .0001) and Midwest (17.4 weeks; P < .0001). Overall, the most common indication for NIPT was AMA-only (n = 710, 48.1%). There was a statistically significant difference ( P < .0002) in the percentage of patients who pursued NIPT because of an indication of AMA only between all 3 locations (62.9% vs 41.6% vs 21.8%). More patients pursued NIPT following an abnormal ultrasound finding in the Midwest (27.1%) compared with the west (9.1%; P < .0002) and east (12.1%; P < .0002). Positive serum screening was a more common indication in the east (18.6%) compared with the west (4.3%; P < .0002) and Midwest (5.7%; P < .0002).
| Demographic | Total | West Coast centers | East Coast centers | Midwest center | West vs East ( P value) | West vs Midwest ( P value) | East vs Midwest ( P value) |
|---|---|---|---|---|---|---|---|
| Number | 1477 | 693 | 522 | 262 | |||
| Maternal age, y | |||||||
| Mean (SD) | 35.1 (5.2) | 36.6 (3.5) | 33.8 (6.1) | 33.5 (5.7) | < .0001 | < .0001 | .5070 |
| Min-max | 14-50 | 17-50 | 14-45 | 14-46 | |||
| Gestational age, wks | |||||||
| Mean (SD) | 15.4 (4.4) | 14.1 (3.4) | 16.1 (4.8) | 17.4 (4.5) | < .0001 | < .0001 | .0003 |
| Min-max | 10-36 | 10-29 | 10-36 | 10-33 | |||
| Indication for NIPT, n (%) | |||||||
| AMA only (age ≥35 y) | 710 (48.1) | 436 (62.9) | 217 (41.6) | 57 (21.8) | < .0002 | < .0002 | < .0002 |
| Previous aneuploidy | 9 (0.6) | 1 (0.1) a | 4 (0.8) a | 4 (1.5) | N/A a | N/A a | N/A a |
| Positive serum screen | 142 (9.6) | 30 (4.3) | 97 (18.6) | 15 (5.7) | < .0002 | .3634 | < .0002 |
| Abnormal ultrasound (includes increased NT) | 197 (13.3) | 63 (9.1) | 63 (12.1) | 71 (27.1) | .092 | < .0002 | < .0002 |
| Other only (ie, maternal anxiety) | 25 (1.7) | 11 (1.6) | 10 (1.9) | 4 (1.5) a | .6636 | .9466 a | .698 a |
| >1, n (%) | 394 (26.7) | 152 (21.9) | 131 (25.1) | 111 (42.3) | .1967 | < .0002 | < .0002 |
| Invasive procedures, n (%) | 98 b (6.6) | 64 c (9.2) | 24 d (4.6) | 10 (3.8) | .002 | .0052 | .6129 |
| CVS, n (%) | 23 (23.5) | 16 (69.6) | 6 (26.1) | 1 (4.3) a | .1336 | .0444 a | .281 a |
| Amniocentesis, n (%) | 75 (76.5) | 48 (64.0) | 18 (24.0) | 9 (12.0) | .0081 | .0422 | .992 |
a n <5, not suitable for the 2-proportion Z-test
b 4 patients had both CVS and amniocentesis
c 1 patient had both CVS and amniocentesis
In total, 98 diagnostic procedures were performed on 94 (6.4%) patients, including 23 CVS and 75 amniocentesis procedures ( Table 1 ). Of these, 64 (65.3%) were performed at the centers in the west ( Tables 1 and 2 ). The 3 most common indications for pursuing an invasive procedure were confirmation following NIPT, fetal abnormality on ultrasound with negative NIPT, and possibility of other genetic abnormality that is not covered by NIPT. Thirty patients with an abnormal result elected an invasive procedure, including 22 of 51 (43.1%) with an aneuploidy detected result and 8 of 41 (19.5%) with an unclassifiable result. There was a statistically significant difference ( P < .0008) between the proportion of procedures performed to confirm an abnormal NIPT result in the west (18 of 31; 58.1%) vs the east and Midwest combined (14 of 61; 23.0%) ( Table 2 ). Four patients underwent both CVS and amniocentesis. In 2 patients, amniocentesis was performed to confirm positive CVS results. One mosaic trisomy 13 by fluorescent in situ hybridization (FISH) with normal culture (NIPT preceding CVS detected aneuploidy for chromosome 13) and a second with normal FISH but 47,+21 on culture (NIPT detected T21). The other 2 patients who underwent both CVS and amniocentesis had negative NIPT results. In 1 patient, both procedure attempts were unsuccessful, and in the second patient the CVS culture did not yield a result but karyotype by amniocentesis was normal. Three procedures were performed following the cancellation of NIPT secondary to excessive cfDNA, all of which resulted in a normal karyotype ( Table 2 ).
| Site | Patients, n | Invasive procedure after abnormal NIPT a | Invasive procedure after negative NIPT | Invasive procedure after cancelled NIPT | Total invasive procedures |
|---|---|---|---|---|---|
| West | |||||
| A | 144 | 5 of 11 (45.5%) | 2 of 130 (1.5%) | 0 of 3 | 7 (4.9%) |
| B | 381 | 11 of 13 (84.6%) | 23 b of 365 (6.3%) | 2 of 3 | 36 (9.4%) b |
| C | 168 | 2 of 7 (28.6%) | 19 of 160 (11.9%) | 0 of 1 | 21 (12.5%) |
| Subtotal | 693 | 18 | 44 | 2 | 64 |
| East | |||||
| D | 78 | 2 of 3 (66.7%) | 3 of 73 (4.1%) | 0 of 8 | 5 (6.4%) |
| F | 444 | 10 c of 35 (28.6%) | 8 of 404 (2.0%) | 1 of 2 | 19 (4.3%) c |
| Subtotal | 522 | 12 | 11 | 1 | 24 |
| Midwest | |||||
| E | 262 | 2 of 23 (8.7%) | 8 b of 231 (3.5%) | 0 of 5 | 10 (3.9%) b |
| Total | 1477 | 32 | 63 | 3 | 98 |
a Includes aneuploidy detected and unclassifiable
b Includes 1 patient who had both CVS and amniocentesis
For 29 patients with an aneuploidy detected NIPT result that did not pursue diagnostic testing, outcome information was provided for 24 (83%). Results are shown in Table 3 . There were 14 live births, 7 pregnancy losses, and 3 elective terminations. One patient with T21 detected by NIPT and preterm delivery showed postnatal karyotype of isochromosome 21 (46,XX,i21[q10;q10]). One preterm delivery with T13 by NIPT died shortly after birth. Two of 7 pregnancy losses had NIPT result confirmed through karyotype on products of conception. All 3 patients who terminated their pregnancy without confirmatory diagnostic testing already showed significant fetal anomalies by ultrasound before blood draw for NIPT (ie, holoprosencephaly, cystic hygroma, ascites). In the case of holoprosencephaly, T13 because of a robertsonian translocation was confirmed by karyotype analysis on cord blood (46,XX,+13, der[13;14][q10;q10]).
| Aneuploidy detected | T21 | T18 | T13 | MX |
|---|---|---|---|---|
| Number | 26 | 10 | 9 | 6 |
| Declined invasive, n (%) | 17 (65.4) | 3 (30.0) | 4 (44.4) | 5 (83.3) |
| Term delivery | 8 a | 1 | 1 | 0 |
| Preterm delivery | 2 b | 0 | 1 | 1 c |
| Pregnancy lost | 5 d | 1 d | 0 | 1 |
| Elective termination | 0 | 0 | 1 e | 2 f |
| Unknown | 2 | 1 | 1 | 1 |
a 7 T21 confirmed by postnatal karyotype
b Includes 1 case of T21 because of isochromosome: 46,XX,i21(q10;q10)
c postnatal karyotype mosaic 47,XXX/45,X
d 1 case confirmed by karyotype on products of conception
e Confirmed T13 due to Robertsonian translocation: 46,XX,+13,der(13;14)(q10;q10)
f Both cases showed cystic hygroma and other abnormalities by fetal ultrasound.
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