Newborn Screening for Cystic Fibrosis in Wisconsin: Comparison of Biochemical and Molecular Methods

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Newborn Screening for Cystic Fibrosis in Wisconsin: Comparison of

Biochemical and Molecular Methods

Ronald G. Gregg, PhD*; Amy Simantel‡; Philip M. Farrell, MD, PhD‡; Rebecca Koscik‡; Michael R. Kosorok, PhD‡; Anita Laxova‡; Ronald Laessig, PhD§; Gary Hoffman§; David Hassemer§;

Elaine H. Mischler, MDi; and Mark Splaingard, MDi

ABSTRACT. Objectives. To evaluate neonatal screen-ing for cystic fibrosis (CF), includscreen-ing study of the screenscreen-ing procedures and characteristics of false-positive infants, over the past 10 years in Wisconsin. An important objective evolving from the original design has been to compare use of a single-tier immunoreactive trypsinogen (IRT) screen-ing method with that of a two-tier method usscreen-ing IRT and analyses of samples for the most common cystic fibrosis transmembrane regulator (CFTR) (DF508) mutation. We also examined the benefit of including up to 10 additional CFTR mutations in the screening protocol.

Methods. From 1985 to 1994, using either the IRT or IRT/DNA protocol, 220 862 and 104 308 neonates, respec-tively, were screened for CF. For the IRT protocol, neo-nates with an IRT 180 ng/mL were considered positive, and the standard sweat chloride test was administered to determine CF status. For the IRT/DNA protocol, samples from the original dried-blood specimen on the Guthrie card of neonates with an IRT 110 ng/mL were tested for the presence of theDF508 CFTR allele, and if the DNA test revealed one or twoDF508 alleles, a sweat test was obtained.

Results. Both screening procedures had very high specificity. The sensitivity tended to be higher with the IRT/DNA protocol, but the differences were not statisti-cally significant. The positive predictive value of the IRT/DNA screening protocol was 15.2% compared with 6.4% if the same samples had been screened by the IRT method. Assessment of the false-positive IRT/DNA pop-ulation revealed that the two-tier method eliminates the disproportionate number of infants with low Apgar scores and also the high prevalence of African-Americans identified previously in our study of newborns with high IRT levels. We found that 55% of DNA-positive CF in-fants were homozygous for DF508 and 40% had one

DF508 allele. Adding analyses for 10 more CFTR muta-tions has only a small effect on the sensitivity but is likely to add significantly to the cost of screening.

Conclusions. Advantages of the IRT/DNA protocol over IRT analysis include improved positive predictive value, reduction of false-positive infants, and more rapid diagnosis with elimination of recall specimens. Pediatrics 1997;99:819 – 824; cystic fibrosis, newborn screening, immunoreactive trypsinogen, population inci-dence, DNA testing.

ABBREVIATIONS. CF, cystic fibrosis; IRT, immunoreactive trypsinogen; DNA, deoxyribonucleic acid.

Cystic fibrosis (CF) is one of the most common autosomal recessive diseases in the Caucasian pop-ulation, with incidence estimates ranging from 1 in 2000 to 1 in 4000.1 A diagnosis of CF is typically

considered because of either a positive family his-tory, neonatal meconium ileus, or the subsequent development of other disease manifestations, eg, fail-ure to thrive, steatorrhea, and/or chronic lung dis-ease. Routinely, the diagnosis is confirmed with a sweat test to demonstrate characteristically high chloride concentration. Unfortunately, there is often a delay in diagnosis.1 Newborn screening for CF

became feasible in 1979 when Crossley and co-work-ers2showed that neonates with the disease have an

elevated immunoreactive trypsinogen (IRT) level. Subsequent studies showed that the IRT assay, when used as either a single test method or with recall (repeat specimen on infants above a designated cut-off level), allowed identification of most asymptom-atic infants with CF.3–5Early detection is potentially

advantageous because of the common delay in diag-nosis and the opportunities screening creates to ini-tiate therapeutic interventions and genetic counsel-ing before the overt presentation of symptoms.6 –9

However, the IRT test has several problems particu-larly in relation to its low positive predictive value. This can be improved by analyzing a second speci-men,1,5but the recall strategy has the drawback that

for many infants in the United States obtaining a follow-up blood sample is not possible. Although only limited information is available, recall specimen loss as high as 22% has been reported for the Colo-rado study,5 however, in an Australian study, loss

attributable to recall was only 2%.10

In 1985, comprehensive evaluation of CF newborn screening in Wisconsin was implemented, both to determine an optimal screening strategy and to de-termine the benefits, risks, and costs of early detec-tion of individuals with CF. The evaluadetec-tion of the clinical implications for early detection is ongoing and is not included in the current report. Assessment of screening procedures in the Wisconsin CF Neona-tal Screening Project has been completed in a two-stage process. First, after four years of screening using a single IRT test, we reported a sensitivity of only 85%,11a value similar to that reported by

oth-From the *Waisman Center for Mental Retardation and Human Develop-ment; ‡Departments of Pediatrics and Biostatistics, §State Laboratory of Hygiene, University of Wisconsin-Madison, Madison, Wisconsin, and the

iMedical College of Wisconsin, Milwaukee, Wisconsin. Received for publication Aug 6, 1996; accepted Oct 15, 1996.


ers.5Lowering the IRT cutoff level to improve

sensi-tivity was explored, but the corresponding increase in false-positive infants, who require sweat tests makes this approach undesirable, especially because the false-positive population includes disproportion-ately high representation of lower risk neonates.12

Consequently, we concluded that “the strategy for cystic fibrosis newborn screening will need to evolve into a true two-tier screening test”.11With

identifica-tion in 1989 of the gene responsible for CF (CFTR) and a mutation,DF508, that accounted for approxi-mately 70% of all mutant CF chromosomes,13 it

be-came possible to add DNA testing as a second tier to the IRT screen. Therefore, after developing molecular genetics procedures for analysis of DF508, using DNA extracts from Guthrie card specimens, we im-plemented an IRT/DNA testing protocol on a pilot basis with a lower IRT cutoff.14 Similar protocols

have been studied sequentially in Australia.10,15 We

now report a concurrent comparison between IRT and IRT/DNA testing protocols and other data from nine years of investigating CF neonatal screening procedures and false-positive infants. In addition, we demonstrate that multimutation testing adds very little when screening typical US populations that have a high frequency of DF508 CFTR and no other mutation at high frequency.


The overall design of the Wisconsin CF neonatal screening project has been described previously.1,11Approval for screening

protocols was obtained from the Human Subjects Committee of the University of Wisconsin and the Research and Publications Committee/Human Rights Board of the Children’s Hospital of Wisconsin. The investigation was designed to include extensive statewide surveillance for new CF patients that might have gone undetected. Two screening strategies, referred to as IRT and IRT/ DNA testing have been compared; in both, the IRT level was determined by radioimmunoassay16(Cis Sorin; Sylmar, CA) using

samples from Guthrie cards typically obtained in the first four days of life (median, 2 days). For the IRT method, implemented on April 15, 1985 and terminated June 30, 1991, those infants with levels$180 ng/mL were considered positive. This IRT cutoff level was selected after reviewing of results from the Colorado pro-gram5 and analyzing approximately 10 000 consecutive

dried-blood specimens to estimate the 99.8 centile. The IRT/DNA pro-tocol was implemented on July 1, 1991 and ended June 30, 1994. Samples from neonates with IRT levels$110 ng/mL were ana-lyzed for the presence of theDF508 CFTR mutation using samples obtained from the original Guthrie card. Those neonates with at least oneDF508 allele were subsequently contacted and CF status determined using the quantitative pilocarpine sweat chloride test as the gold standard diagnostic procedure ($60 mEq/L being positive). For both studies the IRT levels were blinded for half of

the newborn population and this group became a standard diag-nosis or control group; data from this group are not included in the current analyses because the unblinding process will continue until 1998.

Our molecular genetics methods have been summarized pre-viously.14 We obtained denatured DNA from dried-blood

speci-mens on Guthrie cards by first soaking the punched out filter paper circle in methanol and then performing a hot-water extrac-tion. Polyacrylamide gel electrophoresis of PCR-amplified DNA served to identify theDF508 mutation.14Mutations S549N, R553X,

and G551D were screened by PCR amplification of exon 11, fol-lowed by restriction enzyme digests that are diagnostic of each mutation. PCR reactions contained Cetus buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2), 200 mM each dNTP, 1.0mm

each primer (11i-3, 11i-5,17and .4U Taq polymerase) (Perkin-Elmer

Cetus), and either 1-mg genomic DNA or a sample of DNA pre-pared from a Guthrie card.14Reactions were cycled (MJ Research

PTC-100) with an initial denaturation step of 2 min at 95°C, followed by 30 cycles of amplification consisting of 1 min at 94°C, 1 min at 55°C, and 1 min at 72°C, ending with a 5-min soak at 72°C. 10 ml of PCR product used in separate restriction digests containing either MboI, HincII or DdeI. The digested DNA was analyzed on a 10% acrylamide gel, and the bands visualized by silver staining. The gel was soaked in .1% AgNO3(v/v) for 20 min,

rinsed in distilled water, and the bands developed in a solution containing 1.5% NaOH, .15% formaldehyde, and .01% sodium borohydride. Mutations, G542X, W1282X, R117H, R553X, N1303K, 1717–1G3A, R560T, and 62111G3T were analyzed by the ARMS procedure using published primers and conditions.18

A total of 360 patients were studied by multimutation analysis (80% of the Wisconsin CF population). All patients had positive sweat tests and were being followed regularly at one of the state’s two CF centers. DNA was obtained from either Guthrie cards, blood, or cheekbrush samples. DNA was isolated from whole blood using the Puregene DNA isolation Kit (Gentra Systems, Inc). Cheekbrush samples were processed as described.19DNA samples

with known mutations were obtained from Coriell Cell Repository and used as positive controls.


Table 1 provides summary information for the early diagnosis screened group, generated from April 15, 1985 to June 30, 1994. These data represent only half of all births in Wisconsin with the remain-der of the neonates randomized to the standard di-agnosis group. For the IRT method, .17% of all new-borns were above the cutoff, whereas .13% were positive for the IRT/DNA method, and were re-ferred for a sweat test. In the hypertrypsinogenic group of infants the frequency of theDF508 mutation was twice that found in the general population con-firming an observation reported previously.14 The

two screening protocols identified 67 CF patients (Table 1). In addition, 5 patients were identified be-cause of meconium ileus and in the IRT group there were 5 false-negative patients, 3 of whom had CF

TABLE 1. Summary of the IRT and IRT/DNA Screening Methods


Screening period April 15, 1996 to June 30, 1991 July 1, 1991 to June 30, 1994

No. newborns 220 862 104 308

No. with positive screen 369 132‡

No. CF patients

Detected by screen 46 21

False negative with MI 4 1

False negative w/o MI 5

CF patients—total 55 22

CF incidence 1:4015 1:4741

* IRT level$180 ng/mL.


siblings (Table 1). These data also give an overall estimate of the CF incidence (1:4223) in Wisconsin. Consistent with our previous report,14 CF incidence

in Wisconsin is significantly less than the 1 in 2500 frequently quoted.20Although it is remotely possible

that a small number of CF patients have not been ascertained, our surveillance methods lead us to be-lieve that an approximate incidence of 1 in 4000 is more accurate for the total populations of Wisconsin and US newborns.21 This incidence also is similar to

the population incidence in the Colorado study.5

Table 2 summarizes the sensitivity, specificity, and positive predictive values achieved with the two screening protocols. The specificity of the two proto-cols is very high, as with most neonatal screening tests,22 and would not be affected appreciably by

typical variations in the IRT cutoff level.11Sensitivity,

on the other hand, showed considerable variation as in other CF screening studies.5,23The IRT/DNA

pro-tocol tended to yield higher sensitivity because of the lower IRT cutoff value, but statistical analyses did not reveal significant differences compared to the IRT method. Nevertheless, during the 3-year IRT/ DNA screening phase, the sensitivity was 100% ex-cluding patients with meconium ileus (Table 2).

Based on initial data,14 we had expected the

posi-tive predicposi-tive value of the IRT/DNA procedure to be greater than the IRT method. However, the data from the two different time periods show that the positive predictive values for the IRT and IRT/DNA methods are 12.5% and 15.2%, respectively, which are not significantly different. One possible reason for this is that the patient populations are different. To examine this possibility, we extracted those pa-tients that would have been detected by the IRT screen from the IRT/DNA data set. These data are presented in the last row of Table 2. This allows direct comparison of the IRT and IRT/DNA methods using the same neonate population and shows that the positive predictive value of the IRT/DNA method was significantly greater than if the IRT only protocol had been used (15.2% vs 6.4%). The im-provement in positive predictive value using the IRT/DNA method occurs in part because of elimi-nating false-positive infants. We have shown that African-American infants and also neonates with low Apgar scores often have an elevated IRT even though they have a relatively low risk of CF.12Table

3 shows that by comparison with the Wisconsin

new-born population in general, the number of individu-als with a normal Apgar score in the 7 to 10 range is significantly reduced in the false-positive group de-tected with the IRT screening methods, ie, the IRT test tends to identify stressed neonates with low Apgar scores.12In contrast, individuals with a

false-positive IRT/DNA screen have a similar Apgar dis-tribution as the general population.

If the IRT false-positive infants are grouped by race, it can be seen that there is an overrepresentation of African-American infants with respect to their proportion in the general population of Wisconsin newborns. This is of concern because of the relatively lower incidence of CF in the African-American pop-ulation (1 in 11 000217 000),21compared to the

inci-dence of CF in the Caucasian population (1 in 2500

24000).24Breakdown of the false-positive infants for

the two screening methods by race is shown in Table 3. African-Americans represent 27% of the false-pos-itive infants in the IRT screen compared to 5.5% in the IRT/DNA population. The inclusion of the DNA tier to the screen therefore has the added benefit of excluding the overrepresentation of this population of infants, who are at lower risk for CF, from those requiring a sweat chloride test. This advantage of the

TABLE 2. Sensitivity, Specificity, and Positive Predictive Value of the Two Screening Methods

Method (IRT cutoff level) (Dates)

Population Considered Sensitivity (%)

Specificity (%)

Positive Predictive Value (%)

IRT ($180 ng/mL) All 87.0 99.9 12.5

(1985 to 1991)

Asymptomatic (meconeum ileus not included) 90.9 99.9 11.0

IRT/DNA ($110 ng/mL) All 95.2 99.9 15.2

(1991 to 1994)

Asymptomatic (meconium ileus not included) 100 99.9 11.8

IRT$180 (ng/mL) All 85.7 99.8 6.4*

(1991 to 1994)†

* P,05, comparison of data from IRT/DNA (1991 to 1994) data set.

† These data were extracted from the IRT/DNA screen during this period. They represent infants that would have been detected by the IRT only protocol.

TABLE 3. Apgar Score and Race Breakdown for False-Posi-tive Patients

False-Positive Infants Wisconsin†


Apgar at 1 minute n (%) n (%) (%)

0 to 3 14 (10.4) 2 (2.6) 2.8

4 to 6 22 (16.3) 11 (14.5) 8.1

7 to 10 99 (73.3)‡ 63 (82.9) 89.1

Apgar at 5 minutes§

0 to 3 0.0 0 (0) 0.4

4 to 6 10 (7.5) 1 (1.3) 1.2

7 to 10 124 (92.5)‡ 75 (98.7) 98.3


White 326 (61)‡ 102 (91) 87.0

African-American 148 (28)‡ 8 (7) 10.0

Other 61 (11) 2 (2) 3.0

* The data for the IRT cohort includes individuals over the entire study period that had IRT levels$180 ng/mL.

† All Wisconsin births, data from the Center for Health Statistics, Wisconsin Department of Health and Social Services, 1992. ‡ Indicates significantly different from Wisconsin newborn popu-lation (P,.05).


IRT/DNA screening protocol would be magnified in areas in which a large proportion of screened infants are from African-American populations at lower risk for CF.

When a positive IRT/DNA screening result is communicated to the primary care physicians, they frequently wish to know if the IRT level has any predictive value in terms of the likelihood that a particular infant will have CF. Table 4 presents data indicating that neonates with very high IRT levels are considerably more likely to have CF compared to those with low values. Whereas there is a strong correlation, great care and adequate counseling should accompany use of such data in providing risk estimates. It also may be useful in deciding the ur-gency with which the follow-up sweat test, that al-ways is recommended, is obtained. Our protocol re-quired the use of the sweat chloride test to confirm a diagnosis of CF. In infants with two known CFTR mutations a sweat test may not be necessary; how-ever, if this approach is used the DNA test should be repeated on a new sample before indicating the in-fant has CF, to eliminate any possible laboratory errors.

Our current implementation of the IRT/DNA screening protocol only includes screening for the most common CFTR mutation,DF508, as a one mu-tation, second tier test. Our assessment of the 77 CF patients identified in the Wisconsin CF Neonatal Screening Project (Table 1) revealed that 55% had two DF508 alleles and 40% were compound het-erozygotes. One way to improve the screening pro-tocol further would be to increase the percentage of CF chromosomes detected. To examine this possibil-ity we screened samples from 360 patients followed at Wisconsin CF centers for several of the more fre-quent CFTR mutations. Table 5 shows the frequen-cies of these mutations in the Wisconsin population. These frequencies are similar to previously pub-lished reports25and reflect the distribution of CFTR

mutations in the US population of CF patients (Table 6). We also were interested in the impact on the detection percentage of adding either all or some of these mutations to the DNA protocol. The frequency of DF508 is .7125 and therefore, assuming Hardy-Weinberg equilibrium, 91.74% of our CF patients will be expected to carry at least one copy of theDF508

mutation. Table 5 shows that in theory, there is only a 5.5% increase in the detection rate even when all 10 mutations are added to the DNA screen. This would result in detection of one extra CF patient per year in Wisconsin. In view of the increased cost that would be incurred with current methods to add the 10 mutations to the screen it is unlikely that this would be warranted for typical populations in the US. A decision with respect to the mutations used in the second tier would need to consider some potentially hidden costs, such as additional medical costs and legal costs that may be incurred because infants were missed by the screen. It should be noted that short of completely sequencing the CFTR gene in every new-born, some patients will be missed by screening. Further, in populations that have large subpopula-tions with high frequencies of a particular mutation, for example, Ashkenazi Jews (DF508 (27%) and W1282X (51%)),9inclusion of other mutations to the

second tier screen may be warranted. The selection of mutations to use in the screen, based on frequency in the CF population also should recognize that some-times the frequency of a mutation in the general population is different from that in the CF popula-tion.9


Traditionally a diagnosis of CF was made because of either: 1) a positive family history, which occurs in about 20% of cases26,27; 2) the occurrence of

meco-nium ileus; or 3) as a result of either intestinal mal-absorption or chronic pulmonary obstructive dis-ease. Once the characteristic signs and symptoms become evident, the disease can be diagnosed by performing a sweat test using quantitative pilo-TABLE 4. IRT Level as a Predictor of CF in Infants With

Positive IRT/DNA (DF508) Screen*

IRT Level (ng/mL)

No. CF/No. Infants

CF Risk, % (95% CI)

100 to 139 2/1404 0

140 to 179 1/387 0.25 (0 to 0.7)

180 to 219 12/333 3.6 (1.6 to 5.6)

220 to 259 13/122 10.7 (5.2 to 16.2)

260 to 299 11/59 18.6 (8.7 to 28.5)

.300 20/83 24.1 (14.9 to 33.3)

* Estimates are based on observed rates of nonmeconium ileus diagnosis in the IRT and IRT/DNA screened populations. For IRT levels,180 ng/mL, only data from the IRT/DNA group are used. However, all infants in the IRT group (1985 to 1991) have been retroactively tested for the presence ofDF508. Those with either one or two copies of DF508 are included in the $180 ng/mL groups.

TABLE 5. Frequency of CFTR Mutations and Effect of Add-ing Each to CF Detection UsAdd-ing IRT/DNA Test

Mutation Chromosomes* (No. Positive/

No. Tested)

Frequency (%)

Theoretical Cumulative Detection†


Patients Missed in One Year‡

DF508§ 513/720 71.25 91.74 1.32

G542X 17/162 3.02 93.38 1.05

W1282X 7/102 1.97 94.36 0.90

R117H 6/101 1.71 95.14 0.77

R553X 11/197 1.61 95.82 0.66

G551D 9/195 1.33 96.35 0.58

N1303K 3/100 0.86 96.67 0.53

171721G3A 2/99 0.58 96.88 0.49

R560T 2/106 0.54 97.06 0.47

62111G3T 3/163 0.53 97.25 0.44

S549N 0/196 0.0 97.25 0.44

* Chromosomes were analyzed on blood or cheek cell specimens obtained from 360 patients (80% of the total Wisconsin CF popu-lation), all of whom had a positive sweat test.

† Percentage of CF patients expected to carry at least one copy of a detectable mutation. The theoretical cumulative detection rate was calculated assuming Hardy-Weinberg equilibrium (which may not be correct for all mutations and populations) and addi-tion of the mutaaddi-tions to the screen, starting with the most frequent in the Wisconsin population.

‡ Based on expectation that there will be 16 individuals with CF born per year in Wisconsin [70 000 live births 3 1/4400, the predicted relative incidence rate (see Table 1)].


carpine iontophoresis. Unfortunately, the diagnosis of CF is often delayed. Data available from the US Cystic Fibrosis Foundations’s Patient Registry indi-cate that the mean age in 1992 was 4.0 years and the median 1.1 years.1Some studies suggest that delay in

diagnosis is associated with worse disease, including severe malnutrition and irreversible pulmonary in-fections.4,28,29 Other observations, however, do not

support a relationship between age of diagnosis and prognosis.22,30 On the basis of retrospective analysis

of outcomes related to age of diagnosis, Shwachman et al31 recommended newborn screening for CF in

1970. Newborn screening for CF screening became feasible in 1979 when Crossley et al2 applied

mea-surement of IRT to neonatal dried-blood specimens collected on Guthrie cards in New Zealand. They demonstrated that the IRT test could be applied suc-cessfully to such specimens, thereby allowing screen-ing in centralized laboratories. Subsequent experi-ence in other countries was encouraging. In the US, however, the Cystic Fibrosis Foundation recom-mended caution and more research, including criti-cal assessment of the screening procedures and study of the value of early treatment related to prog-nosis; these recommendations were published in 1983 after a Task Force review.32

As a result of nine years of investigation in Wis-consin and elsewhere,10,15an attractive test has been

developed and its validity assessed. The method

in-volves an initial measurement of IRT as a prescreen with a cutoff low enough to detect almost all CF infants without meconium ileus. Advantages of the IRT/DNA test compared with the IRT method in-clude the following. The positive predictive value of the test is increased considerably. Populations at lower risk for CF are preferentially excluded from the screen. Both tiers of the screen use samples from the initial Guthrie card, eliminating the need to col-lect a second sample, which can result in 2 to 20% of the infants being lost to follow-up.5,10

Concurrent with our study, Ranieri et al15and

Wil-cken et al10 have employed a similar strategy. They

report results of adding DNA testing for the DF508 mutation, and Ranieri et al15also investigated adding

three other CFTR mutations (G542X, R553X, and G551D) to the screen. Although in theory the addition of these extra mutations should increase the sensitivity of the screen, in practice all the individuals detected with CF in our study had at least one copy of theDF508 mutation. The positive predictive value for the South Australian study of Ranieri et al15was 31.5%, twice that

for the Wisconsin study. Wilcken et al10reported

posi-tive predicposi-tive values of 13% when one DF508 allele was present and 37.4% overall. The differences are attributable to different IRT cutoff values used in the various studies. In addition, the predictive value of a screening test is dependent not only on sensitivity and specificity but also on characteristics of the population screened, particularly the prevalence of preclinical dis-ease in those tested. Furthermore, the incidence of CF appears to be higher in Australian newborns.10,15,22 In

both Australian studies,10,15 only infants with an IRT

level above the 99th centile were screened for muta-tions compared to the 98th centile (2036/104 308) in our study.

Table 4 shows that 1404 of the 2036 samples posi-tive in the IRT prescreen ($110 mg/mL) had IRT levels below $180 ng/mL; but, there were no CF patients in this group. These data indicate that one can potentially choose a higher IRT level for the first tier cutoff, without decreasing the sensitivity of the screen. It is now clear, however, that there are con-siderable problems with absolute measures of IRT over extended periods of time.16,33Additionally, the

IRT assay seems to exhibit a cyclical season-depen-dent change with a higher number of samples that need to be screened for CFTR mutations in winter compared to summer. Given the temperature ex-tremes in Wisconsin, this is likely to be attributable to the lability of the IRT on the Guthrie card during normal postal transfer to the centralized IRT testing laboratory.33 To avoid these problems, we

recom-mend a protocol which targets a predetermined number of high IRT specimens to be processed for DNA analysis thereby facilitating use of an optimum batch size to reduce costs, as we are currently doing in Wisconsin.34 This means that the absolute IRT

value chosen as the cutoff changes slightly from day to day, but ensures that those samples most likely to be from CF patients are included in the DNA testing. Based on the Wisconsin and Australian experi-ence, it appears that processing the 1% highest IRT specimens (99th centile) is most appropriate. As de-TABLE 6. DNA Analysis of Genotyped CF Patients in the US*

n Percent

DF508 12701 67.7

G542X 403 2.2

G551D 357 1.9

W1282X 240 1.3

N1303K 223 1.2

R553X 157 0.8

3849110kbC3T 102 0.5

62111G3T 147 0.8

171721G3A 101 0.5

R117H 101 0.5

R334W 36 0.2

DI507 42 0.2

R347P 37 0.2

R560T 23 0.1

R1162X 44 0.2

278915G3A 25 0.1

A455E 16 0.1

31201IG3A 14 0.0

S549N 12 0.0

7111IG3T 9 0.0

Other 178 0.9

Unidentified 3814 20.3

Total 18782 99.7†

Patient Genotypes

Allele 1/Allele 2 n % of Genotype

DF508/DF508 4573 48.7

DF508/Known 1511 16.1

DF508/Unknown 2044 21.8

Known/unknown 310 3.3

Known/known 223 2.4

Unknown/unknown 730 7.8

Total 9391 100.0


scribed in detail elsewhere,34 for the routine IRT/

DNA method now being used in Wisconsin, we have lowered the IRT cutoff further (56 ng/mL by the fluoroimmunometric method) and also alert physi-cians when the level is quite high and the DNA (DF508) test is negative. This maximizes the sensitiv-ity, whereas the IRT/DNA method decreases the overall number of false-positive individuals.

Given that our group, Ranieri et al,15and Wilcken

et al10have shown that IRT/DNA testing is superior

to IRT alone, a decision as to the number of muta-tions to be screened for in the second tier must be considered. To obtain complete ascertainment, it would be necessary to include many more than the 10 mutations analyzed in this study. On the other hand, it is unlikely that any nonDF508 mutations would have a frequency high enough to yield many CF patients in Wisconsin or in other typical US pop-ulations. Therefore, if multiple mutations are in-cluded in the screening protocol, careful consider-ation should be given to those that are added, depending on the population characteristics of the region being screened. For example, South Austra-lia15 has a very small Jewish population and,

conse-quently, analysis for the W1282X mutation was not included in the DNA screen. By contrast, this muta-tion was the third most frequent in Wisconsin, and in areas with even larger Jewish populations the omis-sion of this particular mutation would result in de-creased sensitivity of the screen.

We have shown that neonatal screening for CF using a two-tiered IRT/DNA screening protocol has high specificity and sensitivity. In addition, other advantages of the IRT/DNA protocol over IRT anal-ysis include improved positive predictive value, re-duction of known false-positive infants and more rapid diagnosis with elimination of recall specimens.


This research has been supported by grant A001 5– 01 from the Cystic Fibrosis Foundation and grants DK34108 and RR03186 from the National Institutes of Health.


1. Farrell PM, Mischler EH. Newborn screening for cystic fibrosis. Adv

Pediatr. 1992;39:31– 64

2. Crossley JR, Elliott RB, Smith PA. Dried-blood spot screening for cystic fibrosis in the newborn. Lancet. 1979;i:472– 474

3. Heeley AF, Heeley ME, King DN, Kuzemko JA, Walsh MP. Screening for cystic fibrosis by dried blood spot trypsin assay. Arch Dis Child. 1982;57:18 –21

4. Wilcken B, Brown ARD, Urwin R, Brown DA. Cystic fibrosis screening by dried blood spot trypsin assay: results in 75,000 newborn infants.

J Pediatr. 1983;102:383–387

5. Hammond KB, Abman SH, Sokol RJ, Accurso FJ. Efficacy of statewide neonatal screening for cystic fibrosis by assay of trypsinogen concen-trations. N Engl J Med. 1991;325:769 –774

6. Orenstein DM, Boat TF, Stern RC, et al. The effect of early diagnosis and treatment in cystic fibrosis: a seven-year study of 16 siblings. Am J Dis

Child. 1977;131:973–975

7. Wilcken B, Chalmers G. Reduced morbidity in patients with cystic fibrosis detected by neonatal screening. Lancet. 1985;ii:1319 –1321 8. Dankert-Roelse JE, Meerman GJ, Martin A, ten Kate LP, Knol K.

Sur-vival and clinical outcome in patients with cystic fibrosis, with or without neonatal screening. J Pediatr. 1989;114:362–367

9. Kalman YM, Kerem E, Darvasi A, DeMarchi J, Kerem B. Difference in frequencies of the cystic fibrosis alleles deltaF508 and W1282X, between carriers and patients. J Hum Gen. 1994;2:77– 82

10. Wilcken B, Wiley V, Sherry G, Bayliss U. Neonatal screening for cystic fibrosis: a comparison of two strategies for case detection in 1.2 million babies. J Pediatr. 1995;127:965–970

11. Rock MJ, Mischler EH, Farrell PM, et al. Newborn screening for cystic fibrosis is complicated by age-related decline in immunoreactive trypsinogen levels. Pediatrics. 1990;85:1001–1007

12. Rock MJ, Mischler EH, Farrell PM, Bruns WT, Hassemer DJ, Laessig RH. Immunoreactive trypsinogen screening for cystic fibrosis: charac-terization of infants with false-positive screening test. Pediatr Pulmonol. 1989;6:42– 48

13. Kerem B, Rommens JM, Buchanan JA, et al. Identification of the cystic fibrosis gene: genetic analysis. Science. 1989;245:1073–1080

14. Gregg RG, Wilfond BS, Farrell PM, Laxova A, Hassemer D, Mischler EH. Application of DNA analysis in a population-screening program for neonatal diagnosis of cystic fibrosis (CF): comparison of screening protocols. Am J Hum Genet. 1993;52:616 – 626

15. Ranieri E, Lewis BD, Gerace RL, et al. Neonatal screening for cystic fibrosis using immunoreactive trypsinogen and direct gene analysis: four years’ experience. Br Med J. 1994;308:1469 –1472

16. Hassemer DJ, Laessig RH, Hoffman GL, Farrell PM. Laboratory quality control issues related to screening newborns for cystic fibrosis using immunoreactive trypsin. Pediatr Pulmonol. 1991;57:76 – 83

17. Cutting GR, Kasch LM, Rosenstein BJ, et al. A cluster of cystic fibrosis mutations in the first nucleotide-binding fold of the cystic fibrosis conductance regulator protein. Nature. 1990;346:366 –369

18. Ferrie RM, Schwarz MJ, Robertson NH, et al. Development, multiplex-ing, and application of ARMS tests for common mutations in the CFTR gene. Am J Hum Genet. 1992;51:251–262

19. Richards B, Skoletsky J, Shuber AP, et al. Multiplex PCR amplification from the CFTR gene using DNA prepared from buccal brushes/swabs.

Hum Mol Genet. 1993;2:159 –163

20. Boat T, Welsh M, Beaudet AL. Cystic fibrosis. In: Schriver CL, Beaudet AL, Sly WS, Valle D, eds. Metabolic Basis of Inherited Disease. New York, NY: McGraw Hill; 1989:2649 –2680

21. Kosorok MR, Wei WH, Farrell PM. The incidence of cystic fibrosis. Stat

Med. 1996;15:449 – 462

22. Allen DB, Farrell PM. Newborn screening: principles and practice. Adv

Pediatr. 1996;43:231–270

23. Wilcken B. Newborn screening for cystic fibrosis: its evolution and a review of the current situation. Screening. 1993;2:43– 62

24. Kulczycki LL, Schauf V. Cystic fibrosis in blacks in Washington, DC: incidence and characteristics. Am J Dis Child. 1974;127:64 – 67 25. Kazazian HH, Jr. Population variation of common cystic fibrosis

muta-tions. The Cystic Fibrosis Genetic Analysis Consortium. Human

Muta-tion. 1994;4:167–177

26. Blythe SA, Farrell PM. Advances in the diagnosis and management of cystic fibrosis. Clin Chem. 1984;17:277–283

27. Rosenstein BJ, Langbaum TS, Metz SJ. Cystic fibrosis: Diagnostic con-siderations. Johns Hopkins Med J. 1982;150:113–120

28. Mohon R, Wagener J, Abman S, Seltzer W, et al. Relationship of geno-type to early pulmonary function in infants with cystic fibrosis identi-fied through neonatal screening. J Pediatr. 1993;122:550 –555

29. FitzSimmons S. The changing epidemiology of cystic fibrosis. Curr Prob

Pediatr. 1994;24:171–179

30. Kraemer R, Hardon B, Rossi E. Classification at time of diagnosis and subsequent survival of children with cystic fibrosis. Helv Paediatr Acta. 1977;32:107–114

31. Shwachman H, Redmond A, Khaw KT. Studies in cystic fibrosis: Report of 130 patients diagnosed under 3 months of age over a 20-year period.

Pediatrics. 1970;46:335–343

32. Task Force on Neonatal Screening of CF. Neonatal screening for cystic fibrosis: position paper. Pediatrics. 1994;72:741–745

33. Dhondt JL, Farriaux JP. What do immunoreactive trypsin assays mea-sure. Screening. 1994;3:33–38


DOI: 10.1542/peds.99.6.819



Mischler and Mark Splaingard

Kosorok, Anita Laxova, Ronald Laessig, Gary Hoffman, David Hassemer, Elaine H.

Ronald G. Gregg, Amy Simantel, Philip M. Farrell, Rebecca Koscik, Michael R.

and Molecular Methods

Newborn Screening for Cystic Fibrosis in Wisconsin: Comparison of Biochemical


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DOI: 10.1542/peds.99.6.819



Mischler and Mark Splaingard

Kosorok, Anita Laxova, Ronald Laessig, Gary Hoffman, David Hassemer, Elaine H.

Ronald G. Gregg, Amy Simantel, Philip M. Farrell, Rebecca Koscik, Michael R.

and Molecular Methods

Newborn Screening for Cystic Fibrosis in Wisconsin: Comparison of Biochemical

located on the World Wide Web at:

The online version of this article, along with updated information and services, is

by the American Academy of Pediatrics. All rights reserved. Print ISSN: 1073-0397.


TABLE 1.Summary of the IRT and IRT/DNA Screening Methods

TABLE 1.Summary

of the IRT and IRT/DNA Screening Methods p.2
TABLE 3.Apgar Score and Race Breakdown for False-Posi-tive Patients

TABLE 3.Apgar

Score and Race Breakdown for False-Posi-tive Patients p.3
TABLE 2.Sensitivity, Specificity, and Positive Predictive Value of the Two Screening Methods

TABLE 2.Sensitivity,

Specificity, and Positive Predictive Value of the Two Screening Methods p.3
TABLE 5.Frequency of CFTR Mutations and Effect of Add-ing Each to CF Detection Using IRT/DNA Test

TABLE 5.Frequency

of CFTR Mutations and Effect of Add-ing Each to CF Detection Using IRT/DNA Test p.4
TABLE 4.IRT Level as a Predictor of CF in Infants WithPositive IRT/DNA (�F508) Screen*


Level as a Predictor of CF in Infants WithPositive IRT/DNA (�F508) Screen* p.4
TABLE 6.DNA Analysis of Genotyped CF Patients in the US*


Analysis of Genotyped CF Patients in the US* p.5