4.2. IRT/PAP Protocols Detect Less CFSPID
The notion that PAP-based CF NBS protocols detect less CFSPID was primarily based on the fact that the first IRT/PAP protocol by Sarles et al., with its two IRT-dependent PAP cut-off levels, was designed in a way that the majority of CFSPID patients are not detected [8
]. The reason for using this design was based on the assumption that in the IRT range from 50.0 to 99.9 μg/L, lower PAP values could reflect mild CF phenotypes that are not the goal of CF NBS. As expected, those IRT/PAP protocols showed also in the following pilot studies a significantly lower detection rate of newborns with CFSPID [12
]. However, so far, there is no evidence that the PAP concentration generally correlates with the severity of CF disease. This fact is also supported by data from the other pilot studies showing higher PAP concentrations in CFSPID or patients with CFTR
mutations leading to pancreatic sufficiency and low PAP concentrations in some patients with CFTR
mutations leading to pancreatic insufficiency and a severe CF phenotype (e.g., [18
]). When the pilot study on the IRT/PAP strategy was started in Heidelberg in 2008, it was decided that only a single PAP cut-off level of ≥ 1.67 μg/L (before correction of the dilution factor 1.0 µg/L) [11
] should be used. Nevertheless, even with this protocol, a significantly lower detection rate for newborns with CFSPID was found. While only 1.6% of the children positively screened by the IRT/PAP protocol with subsequent detection of 2 CFTR
mutations were newborns with CFSPID, the rate with the IRT/DNA [4
] protocol run in parallel was 7.3% [18
]. These results indicate that a CF NBS with PAP alone can reduce the detection of CFSPID.
4.5. Current PAP-Based CF Screening Protocols in Use
Today, PAP-based CF NS protocols may achieve sufficient performance. One strength of a PAP-based CF NBS is the possibility to use it in multiethnic populations where an appropriate genetic screening is either not possible or is too cost-intensive. Table 1
gives an overview of the performance of PAP-based protocols compared to selected purely biochemical IRT/IRT- or genetic CF NBS protocols. There are currently five European countries where a CF NBS strategy based on PAP is used either in a national or regional setting.
The first country to use PAP at nationwide level after a pilot study [12
] was the Netherlands, which started its national screening program with an IRT/PAP/DNA(35)/EGA protocol in 2011 [16
] (Figure 2
A). The program started using the commercially-available MucoPAP kit (Dynabio, Marseille, France), but, as mentioned above, the photometric measurement was replaced by a flouroimmunoassay during the pilot study. Until 2016, the IRT/PAP part of the protocol was performed as proposed by Sarles et al. [8
], except for the increased IRT cut-off values (now 60 µg/L). However, after the last evaluation published in 2019 [16
], the IRT/PAP part of the screening protocol was changed in two points. Firstly, the lower of the two PAP cut-off values was reduced, and secondly, a safety net was introduced for the PAP step, which is based on the 99.9th IRT percentile, as in the protocol according to Sommerburg et al. [11
]. It may be expected that this variant of the CF-NBS protocol will now have a very high sensitivity and a very good PPV. So far, however, there are no newly-published data on this.
To the best of our knowledge, after the MucoPAP-F became commercially available, it was used for this program. However, it should be noted that in the Netherlands, the two IRT-dependent PAP cut-offs as proposed by Sarles (IRT ≥ 100 µg/L: PAP cut-off ≥ 1.6 µg/L and IRT 60–100 µg/L: PAP cut-off ≥ 3.0 µg/L) were maintained, although it has been recognised that the fluorometric read-out of MucoPAP is higher than that with photometric detection. Nevertheless, this is not a disadvantage for the overall performance. In the genetic part of the protocol, an initial screen will be performed with 35 CFTR
mutations. Following a different procedure in the past, there is, today, a very comprehensive genetic approach (Figure 2
A). All samples showing only 1 CFTR
mutation and those without mutation but with an IRT > 100 (safety net) receive a very high level extensive gene analysis. Nevertheless, the overall sensitivity of the protocol in the evaluated five years is only 90%, which does not meet the criteria of the ECFS standards of care [19
]. The reason for this is clearly the IRT/PAP part and not the DNA (35)/EGA part of the protocol. As shown by Dankert-Roelse et al. 2019 [16
] (given also in Table 1
), seven CF patients were missed by a low IRT and eight by a low PAP. While problems with a low IRT are difficult to circumvent, the majority of CF patients missed by PAP, as described above, might have been found if a protocol like the one according to Sommerburg et al. [11
] or Weidler et al. [14
] had been used.
In Germany, a PAP-based protocol with a DNA analysis as third tier is also used (Figure 2
B). The IRT/PAP-SN part follows the recommendations of Sommerburg et al. 2014, and contains a floating IRT cut-off at the 99.0th percentile and only one PAP cut-off value. Originally, the lower PAP cut-off value (1.6 µg/L) according to Sarles et al. 2005 was used; however, the recommendation is now to apply the 87.5th PAP percentile calculated from PAP values of a nonpreselected population of newborns [25
After the introduction of the new MucoPAP-F-Kit, the PAP cut-off value, e.g., at the CF NBS centre Heidelberg, is 2.1 µg/L.
If a sample is PAP positive, a search for the 31 most common disease-causing CFTR
mutations detected by the German national register will be done. If one or two CFTR mutations are found, the sample is rated CF NBS positive. Also, the IRT-dependent safety net (IRT ≥ 99.9th percentile) is used. While samples whose IRT is between 99.0 and 99.9th percentile will be tested for PAP and DNA, samples with an IRT ≥ 99.9th percentile will be immediately rated CF NBS positive [25
]. As a reason for this decision, the authorities argued that CF patients whose CFTR mutations were not included in the panel should not be discriminated on the basis of their origin. The expected PPV was calculated in a post hoc analysis and was expected to be 20%, which would not meet the European standards of care [19
]. This kind of IRT-dependent safety net remains questionable also for other reasons. For example, there is currently no modern CF NBS protocol in which a sample is considered positive after an ultra-high IRT alone. Furthermore, it was shown that, as previously expected, only about 25% of CF patients diagnosed with this protocol received a search for CFTR
mutations during the CF NBS protocol [26
]. Based on data from the Heidelberg IRT/PAP+SN pilot study, the sensitivity of the protocol was estimated to be 96% in the post hoc analysis mentioned above [26
]. A complete evaluation of the CF NBS protocol used in Germany is now scheduled to be conducted after 3 years of application.
Portugal started in 2016 with an IRT/PAP-SN/IRT protocol which was evaluated before in the aforementioned pilot study (Figure 2
]. To the best of our knowledge, there are currently no changes in the protocol. The IRT cut-off level was set at 65 µg/L. PAP is measured with the Muco PAP F kit. The PAP cut-off values are IRT dependent: If the IRT value is between 65 and 100 µg/L, a PAP cut-off value of ≥ 1.6 µg/L applies, with an IRT value of ≥ 100 µg/L a PAP cut-off value of ≥ 0.5 applies. Furthermore, an IRT SN strategy (≥150 µg/L) also triggers the measurement of a second IRT (50 µg/L). In our opinion, the PAP cut-off values seem rather low considering the fluorimetric readout of the MucoPAP-F kit used. However, this approach may be advantageous for the sensitivity of the protocol with regard to the multiethnic population in Portugal, especially since the second IRT measurement in IRT/PAP positive neonates will achieve a PPV as required by the European standards. In the pilot study the sensitivity was 94.4% and the PPV 41.03% [17
In 2017, Austria changed from an IRT/IRT to an IRT/PAP-SN/IRT protocol. PAP measurement is done with the MucoPAP II kit. For the initial IRT, a cut-off value of 65 ng/L was set. The PAP measurement is based on Sarles et al. with two IRT-dependent PAP cut-off values [8
] that were adapted to the conditions of MucoPAP II: If IRT is between 65 and 100 µg/L, a PAP cut-off value of ≥ 2.5 µg/L applies, if IRT is ≥ 100 µg/L, a PAP cut-off value of ≥ 1.33 µg/L is valid. In addition an IRT-dependent SN (IRT ≥ 130 µg/L) is used. Both an increased PAP and an ultra-high IRT (SN) trigger the second IRT (sampled after 3–4 weeks of age, cut-off value 50 µg/L) [27
In 2018, Catalonia started using an IRT/PAP-SN/IRT/DNA strategy. PAP measurement is done with the MucoPAP-F kit. The initial IRT cut-off value was set at 50 ng/L. For the second tier, two IRT-dependent PAP cut-off values [8
] are used, but with other cut-off values, as published elsewhere: If IRT is between 50 and 80 µg/L, a PAP cut-off value of ≥ 1.95 µg/L is used, if IRT is ≥ 80 µg/L, a PAP cut-off value of ≥ 1.0 µg/L applies. An IRT dependent SN with an IRT cut-off value of ≥ 130 µg/L was also implemented in Catalonia. Both an increased PAP and an ultra-high IRT (SN) trigger the second IRT (sampled after 21–30 days of life, IRT cut-off value 35 µg/L). If the second IRT is positive, a comprehensive genetic analysis is performed [28
Of the PAP-based CF NBS protocols currently used in a national or regional screening programme, only the Netherlands has so far provided performance data of sufficient quality [16
]. It is obvious that the data from the other programmes must also be evaluated without delay and the results published. PAP-based protocols definitely have advantages in multiethnic populations, and help to detect less carriers and CFSPID. While the problem of a too low PPV caused by purely biochemical IRT/PAP protocols is probably no longer relevant, as currently, only protocols with at least three tiers are in use, the problem of sufficient sensitivity remains of high relevance.