2. The Evolution of the PAP Kit
3. Description of Selected European Pilot Studies
4. Findings from the Pilot Studies
4.1. IRT/PAP Protocols Detect Less Healthy Carriers
4.2. IRT/PAP Protocols Detect Less CFSPID
4.3. IRT/PAP Protocols May Show Lower Sensitivity than IRT/DNA Protocols
- The use of an IRT-dependent safety net: When the pilot studies were started in the Germany, the general concern was that the PAP strategy had a worse sensitivity than a well-performing genetic CF NBS. Similar to the IRT/DNA protocols with a restricted mutation panel, an IRT-dependent safety net was added six months after starting the pilot studies. Therefore, CF NBS is considered positive if the initial IRT is above the 99.9th percentile, regardless of the PAP result. When the results of the pilot study conducted in Prague (Czech Republic) were published in 2012, the IRT/PAP strategy showed a very low sensitivity of only 76% . After a re-evaluation for a joint, posthoc analysis of the raw data from Prague, Dresden and Heidelberg, it was found that the sensitivity of the Prague PAP-based CF NBS would have been 89.5% if the colleagues there had used the original IRT/PAP protocol but with the IRT-dependent safety net, as was done in the German centres . Furthermore, a recently published paper on the Dutch CF NBS shows that out of eight CF patients not detected in the IRT/PAP part of the IRT/PAP/DNA(35)/EGA strategy, five would probably have been found if such an IRT-dependent safety net had been used .
- Renouncing the two IRT-dependent PAP cut-off values: As mentioned, the reason to use the two IRT-dependent PAP cut-offs was based on the assumption that such a protocol would avoid the detection of CFSPID. In addition, IRT/PAP protocols with two IRT-dependent PAP cut-off values were proposed to detect less healthy newborns as false positives compared to protocols with only one PAP cut-off value. However, the results of the aforementioned joint posthoc analysis of the data from Prague, Dresden, and Heidelberg suggest that IRT/PAP protocols with two IRT-dependent PAP cut-off values may have limited sensitivity compared to those with only one PAP cut-off value. In a joint simulation of raw data from Prague and Heidelberg, it was found that by using two PAP cut-off values, four newborns with two mutations in the CFTR gene would have been missed, but would have been detected by the protocol with one PAP cut-off. Only one out of these four newborns carried a CFTR mutation with varying clinical consequence and had a normal sweat chloride. The other three newborns were diagnosed with classical CF with pancreatic insufficiency. Two out of these three CF patients suffered from MI and would have been diagnosed clinically. However, the third CF patient would have been missed by all IRT/PAP protocols relying on two IRT-dependent PAP cut-offs . It can be argued whether one has to consider three missed patients with CF or only one, since two out of these three presented with MI.
- Anyway, the fact that newborns carrying two CF-causing mutations were not detected due to the IRT/PAP protocol with two PAP cut-offs raises the question of whether such a protocol can achieve sufficient sensitivity. It is interesting to note that if the colleagues in Prague had used the same IRT/PAP protocol as that used in Heidelberg, not only with the IRT dependent safety net, but also with only one PAP cut-off value, the sensitivity would have been 94.7%. Also, in a recently published work on the aforementioned Dutch CF NBS program, it was shown that if only one PAP cut-off value had been used, one CF patient out of the eight CF patients not found would still have been detected. With the five CF patients that would have been found by the safety Net, six of the eight CF patients would have been found .
- Lowering of PAP cut-off values: Due to the fact that all the pilot studies mentioned above were started with a MucoPAP kit whose PAP cut-off values had not yet been sufficiently evaluated, the most obvious solution for sensitivity problems would have been to simply adjust the PAP cut-off values downwards. Actually, this was also done later by Sarles et al. and reported in a publication in 2014 . However, significantly lowered PAP cut-off values were not only found there, but were seen in recent years also in other PAP-based protocols (e.g., ). Yet, it is precisely this approach that significantly increases the number of false-positive newborns detected.
- Using both biochemical markers, IRT and PAP, at the same time: In all current PAP-based CF-NBS protocols, IRT and PAP are used sequentially. However, the simultaneous use of both biomarkers instead of two steps, e.g., by using the product of IRT and PAP, has the potential to make the screening strategy significantly more sensitive than in the IRT/PAP protocols currently in use. Despite the simultaneous use of both parameters, IRT can still be used as a first-tier-parameter that triggers the PAP measurement if it is above a certain cut-off value. Such an approach was demonstrated by the Dresden group in a posthoc analysis using raw data from the pilot studies of the two German CF NBS centres, i.e., Dresden and Heidelberg . The data from Heidelberg showed the highest sensitivity with the IRTxPAP product (98.3%), in contrast to the revised strategy of Sarles et al. published in 2014 (94.9%), and also in contrast to the Heidelberg IRT/PAP-SN protocol (96.6%).
- Time-dependent sampling of the dried blood for neonatal screening: There is unpublished local experience from Australia, still acknowledged by a number of CF NBS specialists, that the use of PAP is not sensitive enough if the dried blood sample for NBS is taken from the infant before the age of 48 h. As a reason for this, it was assumed that the PAP blood levels in infants with cystic fibrosis increase over time. According to our experience, this could be true, but not only in CF infants. In Germany, the collection of the dried blood sample is usually carried out between the 36th and 72nd hour of life, but for special reasons, we sometimes see early or late sampling. If we group all available PAP values of the infants studied in recent years into 12-h intervals, we see a trend of an increase in the 25th, 50th, and 75th percentiles from 24 h to 72 h (personal communication O. Sommerburg). However, when we focused on CF patients not found in our IRT/PAP protocol, we could not confirm that these CF patients were missed because the time of collection of the dry blood sample was before the 48th hour of life. In this regard, after more than 10 years of PAP-based CF NBS, we consider it to be proven that PAP screening with samples collected between 36 and 48 h of life is feasible. Yet, if the majority of infants in a country are screened for NBS before the 36th hour of life, we might imagine that PAP blood levels might still be too low. In this case, we would recommend a comprehensive pilot study to test the feasibility of a PAP-based CF NBS also under these conditions.
4.4. Pure Biochemical IRT/PAP Protocols Show a Relatively Low Positive Predictive Value
4.5. Current PAP-Based CF Screening Protocols in Use
Conflicts of Interest
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|2nd Tier Test||Reference||Protocol||Region/Country||n Screened||Prevalence of CF||Sensitivity (%) w/o MI||PPV (%)|
|ECFS standard ||≥95||≥30|
|IRT||Calvin et al. 2012 ||IRT/IRT||East Anglia (UK)||582,966||1:2286||93.8||67.3|
|DNA||Calvin et al. 2012 ||IRT/DNA(29)/IRT||East Anglia (UK)||147,764||1:2111||90.2||85.9|
|Sommerburg et al. 2015 ||IRT/DNA(4)+SN||Southwest Germany||252,020||1:4582||95.1||15.3|
|Kharrazi et al. 2015 ||IRT/DNA(28–40)/EGA||California||2,573,293||1:6899||92||34|
|Sontag et al. 2016 ||IRT/IRT/DNA(41–48)||Colorado, Wyoming, Texas||1,520,079||1:5548||96.2||19.7|
|Lundman et al. 2016 ||IRT/DNA/EGA||Norway||181,859||1:8660||95||43|
|Skov et al. ||IRT/DNA(1)/EGA||Denmark||126,338||1:4866||91.7||84.6|
|PAP||Sommerburg et al. 2015 ||IRT/PAP+SN||Southwest Germany and East-Saxony (Germany)||328,176||1:4860||96.0||8.8|
|Weidler et al. ||IRTxPAP||Southwest Germany and East-Saxony (Germany)||410,111||1:5258||97.4||8.2|
|Marcao et al. 2018 ||IRT/PAP/IRT||Portugal||255,000||1:7500||94.4||41.3|
|Dankert-Roelse et al. 2019 ||IRT/PAP/DNA(35)/EGA||The Netherlands||819,879||1:6029||90||63|
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