Introduction
In many countries, prenatal diagnosis by chorionic villus sampling or amniocentesis is routinely offered to pregnant women who have an increased risk of carrying a child with a chromosomal abnormality. Amniocentesis is the most commonly used invasive prenatal diagnostic procedure in western countries and is performed in about one in 30 pregnancies [
1,
2].
Karyotyping is considered the reference test to detect fetal genetic abnormalities in amniotic fluid cells with considerable accuracy [
3,
4]. However, it is labor-intensive and the costs are high. Furthermore, obtaining results takes 2–3 weeks and the extensive detection capacity of karyotyping can be perceived as a disadvantage due to the detection of abnormalities with unclear or mild clinical relevance, causing difficult counseling issues, patient anxiety, emotional dilemmas concerning the continuation of pregnancy and, albeit rare, unwarranted pregnancy terminations [
5,
6].
Due to these disadvantages, karyotyping as a routine test has been challenged for relatively low-risk indications. In 2003, a molecular PCR-based technique, multiplex ligation-dependent probe amplification (MLPA) became available to detect fetal aneuploidies in amniotic fluid cells [
7]. Following the results of preclinical laboratory studies, MLPA has been proposed as a promising alternative for the detection of the most common chromosomal aneuploidies, i.e. trisomy 13, 18, 21 and sex chromosome aneuploidies. Compared to karyotyping, MLPA has several potential advantages; the waiting time for test results is reduced with simultaneous reduction of anxiety, the preceding prenatal counseling process can focus on the most common chromosomal aneuploidies, and the technique is considerably less labor-intensive and more suitable for high-throughput testing, thereby exploiting economies of scale.
Nowadays, much effort has been put into priority setting based on a trade-off of costs and health gains. From an economic perspective, the preferred prenatal diagnostic strategy is the one that overall yields favorable health gains relative to associated cost differences [
8].
In order to compare the MLPA and karyotyping strategies in terms of diagnostic accuracy, health-related quality of life and cost-effectiveness, we initiated a prospective diagnostic study comparing MLPA with karyotyping in routine clinical practice; the MLPA And Karyotyping, an Evaluation (MAKE) study (ISRCTN47252164) [
9]. If MLPA has comparable diagnostic accuracy and is able to reduce maternal anxiety and costs in routine clinical practice, MLPA could present a suitable substitute for karyotyping. Our research question was: what are the costs and effects of MLPA compared to karyotyping when applied to the indications advanced maternal age, increased risk following prenatal screening and anxiety?
Results
Patients and test results
Between March 2007 and October 2008 we included 4,585 consecutively pregnant women. Patient and procedural characteristics are displayed in Table
1. In 4,484/4,585 samples (97.8%) MLPA and karyotyping were concordant, showing normal results in 4,387/4,585 (95.7%) and aneuploidy in 98/4,585 (2.1%). Discordant results were found in 26/4,585 (0.6%) samples, representing an abnormal karyotype undetected by MLPA. All aneuploidies of chromosomes 13, 18, 21 and non-mosaic X and Y were also detected by MLPA. MLPA, by design, could not detect three severe chromosomal abnormalities other than trisomies 13, 18, 21. In 75 cases (1.6%) the MLPA test result failed. Karyotyping failed in one of these 75 cases (0.02%). Diagnostic accuracy of MLPA was comparable (non-inferior) to karyotyping (1.0 95% CI 0.999–1.0). Sensitivity and specificity for the detection of trisomies 13, 18, 21, X and Y were 100% (95% CI 96–100%) and 100% (95% CI 99.9–100%), respectively [
11]. There were neither statistical nor clinically relevant differences in HRQoL. Summary physical and mental health scores between people receiving a karyotype or MLPA did not differ (mean difference 0.31; 95% CI −3.06 to 2.44;
p 0.82 and mean difference 1.91 95% CI −1.15 to 4.99;
p 0.22, respectively) (see Table S1). Therefore, we considered cost-minimization analysis the appropriate framework.
Table 1
Baseline and procedural characteristics
Indication (%) |
Advanced maternal age | 3,464 (75.6) |
Increased risk following prenatal screening | 1,074 (23.4) |
Anxiety | 47 (1.0) |
Median gravidity (5th to 95th%) | 2 (1 to 5) |
Median parity (5th to 95th%) | 1 (0 to 3) |
Median gestational age (weeks + days) (5th to 95th%) | 16 +1 (14 + 6 to 17 + 4) |
Withdrawn amniotic fluid (median) (5th to 95th%) | 20 ml (16.0 to 20.0) |
Color of amniotic fluid |
Clear/yellow | 4,467 (97.4%) |
Red/brown/turbid/green | 118 (2.6%) |
Cell pellet color |
White | 3,923 (85.6%) |
Trace of blood | 381 (8.3%) |
Red/brown/green, yellow, turbid | 281 (6.1%) |
Amniotic fluid for MLPA (median) (5th to 95th%) | 4 ml (2.0 to 8.0) |
Short-term costs
The test costs of the MLPA test performed in duplicate were €344,60 per sample (65% direct and 35% overhead costs), while the costs of karyotyping was €668,00 per sample (74% direct and 26% overhead costs). Table
2 details the volumes of resource use, unit costs per resource and the total short-term costs.
Table 2
Short-term costs: resource use and costs between amniocentesis and the decision to continue or terminate pregnancy
Direct costs in the hospital
|
Primary procedure |
MLPA | 4,585 | 87.3% | 0 | 0.0% | €344.60 |
Karyotyping | 0 | 0.0% | 4,585 | 93.98% | €668.00 |
Additional diagnostic tests |
Karyotyping | 173 | 6.4% | 0 | 0.0% | €668.00 |
FISH | 5 | 0.22% | 11 | 0.27% | €809.00 |
Additional diagnostic tests in case of CA |
Parental karyotyping | 0 | 0.0% | 44 | 0.90% | €668.00 |
DNA and/or biochemical investigation | 0 | 0.0% | 3 | 0.09% | €934.00 |
Ultrasound examination (type II) | 22 | 0.79% | 34 | 0.68% | €653.00 |
Outpatients visit in case of CA |
Consult gynecologists (2 visits) | 35 | 0.48% | 35 | 0.26% | €246.00 |
Consult clinical geneticist and social worker (2 visits) | 63 | 4.82% | 89 | 3.78% | €1,385.00 |
Direct medical costs outside the hospital
|
Travel costs in case of CA (2 visits to hospital) | 98 | 0.05% | 124 | 0.04% | €9.48 |
Total short-term costs per sample | | €394.93 | | €710.65 | |
In the MLPA strategy, 173 subsequent karyotyping were performed because MLPA failed (n = 75) or because MLPA showed a chromosomal abnormality and inheritance patterns needed to be examined (n = 98). Repeat amniocentesis did not occur. MLPA was repeated in 1.6% (5th to 95th percentile 1.3–2.1%) due to an inconclusive result. Five subsequent FISH analyses were done: three because MLPA showed a deletion on a single probe and the laboratory wished to exclude a sub-microscopic deletion, and two for a mosaic chromosome pattern (combined mosaic pattern of Turner and Down syndrome and a mosaic pattern for Turner syndrome and a normal female cell line). In 22 cases in the MLPA strategy and 34 cases in the karyotyping strategy, advanced ultrasound examination was required to exclude other severe congenital abnormalities (e.g. cardiac abnormalities) in the presence of the chromosomal abnormality to support the decision to continue or terminate the pregnancy.
In the karyotyping strategy, 11 subsequent FISH analysis were performed for various reasons; additional information on the grade of mosaicism (mosaic pattern Turner and Down syndrome, mosaic pattern of Turner syndrome) (n = 2), for marker chromosomes (n = 4), de novo unbalanced chromosomal abnormalities (n = 2), a chromosomal abnormality which appeared to be a normal variant (n = 2), and for a mosaic pattern of male and female karyotype which was determined to be a culture artefact (n = 1). In the latter case, biochemical investigation on amniotic fluid was also carried out to determine the testosterone/FSH ratio and karyotyping was repeated in a postnatal sample. In 24 cases parental karyotyping was performed to address the origin of the chromosomal abnormality (inherited or de novo). To assess the consequences of the de novo interstitial deletion, MLPA on subtelomeres and a genomic micro array was carried out. One karyotype failed due to contaminated amniotic fluid (blood and clots). Repeat amniocentesis was offered but the prospective parents declined.
The median short-term costs per sample, i.e. from amniocentesis until the decision to continue or terminate pregnancy, were €344.60 (range €344.60–3.216.08) for the MLPA strategy and €668.00 (range €668.00–4.669.48) for the karyotyping strategy. The short-term costs of the MLPA strategy were on average €315.68 (bootstrap 95% CI €315.63–315.74) lower than the karyotyping strategy (−44.4%).
Long-term and total costs
Table
3 displays the main volumes of resource use, the unit costs per resource use and the long-term costs following the decision to continue or terminate pregnancy. Seventy-six pregnancies in the MLPA strategy (72 clinically severe, 4 clinically uncertain, 0 clinically not relevant) and 79 pregnancies in the karyotyping strategy (74 clinically severe, 5 clinically uncertain, 0 clinically not relevant) were terminated (Table S2). In two pregnancies, postnatal karyotyping was carried out to confirm the prenatal diagnosis (mosaic marker chromosome and mosaic Turner).
Table 3
Long-term costs: resource use and costs after the decision to terminate or continue pregnancy
Terminated pregnancies for detected CA |
Total termination of pregnancy | 76 | 2.2% | 79 | 2.5% | €1,314,00 |
Clinically severe CA (T21/13/18 and other) | 72 | | 74 | | – |
Clinically uncertain CA (X/Y and other) | 4 | | 5 | | – |
Travel costs | 76 | 0.01% | 79 | 0.01% | €4,74 |
Productivity loss for terminated pregnancies with CA | 76 | 11.2% | 79 | 12.9% | €6,730,38 |
Continued pregnancies for detected CA |
Clinically severe CA (T21/13/18 and other) | 13 | 56.8% | 14 | 68.1% | €200.000,- |
Clinically uncertain CA (X/Y and other) | 9 | 2.4% | 14 | 4.1% | €12.000,- |
Clinically not relevant CA (other) | 0 | 0.0% | 17 | 0.0% | – |
Confirmation of prenatal cytogenetic result after birth | 0 | 0.0% | 2 | 0.04% | €739,72 |
Productivity loss for continued pregnancies with severe CA | 13 | 9.6% | 14 | 11.6% | €33.940,- |
Productivity loss for continued pregnancies with uncertain CA | 9 | 0.4% | 14 | 0.7% | €2,036,40 |
Productivity loss for continued pregnancies with not relevant CA | 0 | 0.0% | 17 | 0.0% | – |
Costs for undetected chromosomal abnormalities |
Other clinically severe CA | 3 | 13.1% | 0 | 0.0% | €200.000,- |
Other clinically uncertain CA | 6 | 1.6% | 0 | 0.0% | €12.000,- |
Other clinically not relevant CA | 17 | 0.0% | 0 | 0.0% | – |
Productivity loss for undetected severe CA | 3 | 2.4% | 0 | 0.0% | €36.861,- |
Productivity loss for undetected uncertain CA | 6 | 0.3% | 0 | 0.0% | €2.211,66 |
Productivity loss for undetected not relevant CA | 17 | 0.0% | 0 | 0.0% | €0,00 |
Total long-term costs per sample | | €997,85 | | €896,19 | |
The median long-term costs per sample, i.e. from the decision to continue or terminate pregnancy onwards, were €0.00 (range €0.00–233,940.00) for the MLPA strategy and €0.00 (range €0.00 to 237,000.08) for the karyotyping strategy (Table
2). The long-term costs of the MLPA strategy were on average €76.42 higher compared to the karyotyping strategy (bootstrap 95% CI €71.32–81.52; +8.6%) per sample.
The total costs, including both short- and long-term costs, were median €344.60 (range €344.60–237,000.08) for the MLPA strategy and €668.00 (range €668.00–238,956.48) for the karyotyping strategy. The total cost difference was €240.13 (bootstrap 95% CI €235.02–€245.23) in favor of MLPA (cost reduction −14.9%).
Sensitivity analysis
Table
4 displays the results of the sensitivity analyses. Total MLPA costs were sensitive to the following parameters: the proportion of women deciding to terminate pregnancy, women allowed individual choice, the level of sample throughput, and performing both MLPA and karyotyping. Except for the combined MLPA and karyotyping strategy, the total costs difference remained in favor of MLPA.
Table 4
Sensitivity analysis: impact of parameters varied on short-term, long-term and total MLPA costs per sample (US dollars, %) compared to baseline; and impact on the total (short term and long term) cost difference of MLPA and karyotyping
Baseline strategy | 394.93 (n.a.) | 997.85 (n.a.) | n.a. |
Failure rate |
1.31% | 392.75 (−0.6%) | 997.85 (−) | −2.19 (−0.2%) |
2.05% | 397.70 (+0.7%) | 997.85 (−) | +2.77 (+0.2%) |
Only samples with clear amniotic fluid analyzed with MLPA | 401.44 (+1.7%) | 997.85 (−) | +6.50 (+0.5%) |
% TOP if severe CA |
80% | 394.93 (−) | 1,195.18 (+20.8%) | +197.33 (+14.2%) |
70% | 394.93 (−) | 1,638.32 (+64.2%) | +640.48 (+48.7%) |
Women are allowed individual choice | 552.18 (+39.8%) | 971.65 (−2.8%) | +131.05 (+9.4%) |
Sample throughput |
286 samples/year | 500.55 (+26.7%) | 997.85 (−) | +105.62 (+7.6%) |
1,153 samples/year | 374.94 (−5.1%) | 997.85 (−) | −19.99 (−1.4%) |
One nationwide MLPA laboratory | 294.68 (−29.6%) | 997.85 (−) | −100.25 (−7.20%) |
All samples analyzed with MLPA and karyotyping | 660.85 (+167.3%) | 896.19 (−10.1%) | +599.19 (+43.02) |
Discussion
We evaluated the cost-effectiveness of two prenatal diagnostic test strategies: MLPA and karyotyping. Diagnostic accuracy of MLPA was comparable to karyotyping and health-related quality of life was equal between strategies. For the complete testing process, the MLPA strategy lead to a 14.9% cost reduction per amniotic fluid sample for women with relatively low-risk indications (−44.4% on the short and +8.6% on the long term).
Our study has several limitations. Firstly, we used the data of the nationwide prospective MAKE study which prioritized karyotyping, since at least 12 ml of amniotic fluid was required. The failure rate of MLPA (1.6%) may be lower when MLPA is applied as stand-alone technique since MLPA requires at least 1–2 ml. Sensitivity analysis, however, showed that variations in failure rate had little impact on the overall cost difference. Secondly, we were unable to measure quality of life for women who decided to continue or terminate pregnancy in case of a chromosomal abnormality and in parents with rare prenatally undetected fetal chromosomal abnormalities. However, diagnostic accuracy was high and comparable; we can speculate that the decision to continue or terminate pregnancy in case of a chromosomal abnormality is the same, regardless of the diagnostic test used. The three severe chromosomal abnormalities undetected by MLPA may result in a decreased quality of life at the individual level but not in differences at the group level. Thirdly, we did not adjust the costs associated with pre-test counseling. We expect that targeted testing reduces complex counseling issues and is therefore less costly. Furthermore, three FISH analysis were done following MLPA to exclude a sub-microscopic deletion. If MLPA is implemented as a stand-alone test to detect trisomies 13, 18, 21 X and Y, our research group advises to neglect the interpretation of the quantification of single probes. Taking this into account, the cost reduction of MLPA compared to karyotyping may be even larger than we estimated. Finally, costs were based on Dutch health care costs, which might differ from costs in other countries. That does not imply that the outcome of MLPA being the strategy with lowest costs is not externally valid for other countries. Hence, overall cost differences between countries do affect the absolute cost differences, but do not affect the ordering of strategies in terms of costs.
Compared to other RAD techniques, MLPA and quantitative fluorescent polymerase chain reaction (QF-PCR) are both suitable techniques for high-throughput testing at lower costs compared to fluorescent in situ hybridization (FISH) [
26]. A cost analysis of QF-PCR and revealed that both tests are sensitive to sample throughput and staff skill-mix FISH (2003) [
26]. For laboratories with a throughput of 1,000 samples per annum, karyotyping is the most expensive test to perform, with FISH and Q-PCR calculated to incur approximately half the direct test costs of karyotyping [
26]. However, these studies did not include long-term costs. Due to differences in methodology, a full comparison with our study is impossible.
Costs differences were insensitive to variations in failure rate, or using MLPA on contaminated amniotic fluid samples. However, the costs of MLPA proved sensitive to the proportion of terminated pregnancies and therefore to societal trends, but this is unlikely to affect the overall cost difference. Furthermore, the costs of the MLPA strategy were sensitive to sample throughput as well as the concentration of MLPA analyses in one nationwide centre. This shows that the costs and cost differences depend on the way care is organized. Since the impact of concentration on costs was larger than the impact of higher throughput, we recommend the use of one (or several) nationwide MLPA laboratories. Our study also shows that a combined strategy of MLPA followed by karyotyping is rather inefficient. Costs are considerably increased without any gain in diagnostic accuracy or health-related quality of life compared to the karyotyping only strategy.
The provision of a rapid, unambiguous and a low cost result is an incentive to implement MLPA. Successful implementation, however, also requires the support of pregnant women. If one supports individualized choice for principle or other reasons [
27], one could argue that the decision to either obtain as much cytogenetic information as possible versus a rapid but specific result on the most common chromosomal abnormalities is most appropriately made by individuals who will bear the responsibility for raising the child. Our study shows that allowing individualized choice—assuming that 50% chooses karyotyping and 50% chooses MLPA [
23]—also has large impact on costs, reducing the cost difference of €240 per sample to €83 per sample). While individual choice as strategy is less efficient than a uniform strategy in which every patient would receive MLPA, the overall cost reduction is still in favor of MLPA over the current karyotyping strategy. One could argue that offering a choice between the tests meets most individual needs and wishes, and thereby might outweigh the cost difference. In a discrete choice experimentation [
28], women valued the comprehensive information of karyotyping at £791 and the simple and quick information of a Down only test at £690. This supports our idea that the option to choose may outweigh the previously mentioned efficiency loss of €240 per sample to €83 per sample.
Conclusion
In summary, MLPA is able to detect trisomies 13, 18, 21, X and Y with comparable diagnostic accuracy [
10,
29,
30] and without adverse effect on quality of life at considerably lower costs for the complete testing process. We conclude that MLPA is the preferred strategy and recommend substitution of karyotyping for MLPA for relatively low-risk indications. Future research should be done to evaluate which RAD technique delivers best ‘value for money’, to estimate the cost-effectiveness of this RAD technique on chorionic villus biopsy, and to evaluate the most advantageous organization for the optimal RAD technique.
This study was approved by the medical review ethics committee of the Onze Lieve Vrouwe Gasthuis (reference number 06032).
Acknowledgments
This study was supported by an unconditional grant 945-27-047 from ZonMW, The Netherlands Organization for Health Research and Development, The Hague, The Netherlands.