n terms of screening, diagnostic testing, and treatment, the response to cystic fibrosis (CF) in the U.S. has improved markedly during the past two decades. All states now screen newborns for CF, and the predicted median age of survival for a person with CF is now in the late 30s. At the same time, potential errors in screening remain. This issue of Strategies explores a recent study that underscores how CF screening can continue to improve.

In the late 1990s, the U.S. made rapid progress in newborn screening for CF, going from only three states performing CF neonatal screening in 1994, to all 50 states and the District of Columbia by 2010. However, a missed or delayed diagnosis of CF after newborn screening still can occur, due to a number of causes. A recent paper enumerates 14 such factors, illustrated with six cases of mishaps throughout the screening process drawn from the Wisconsin and Oregon public health laboratories (Pediatr Pulmonol 2011 Dec;46:1166-1174).

According to lead author Michael Rock, MD, the study was intended to make clinicians more aware of how CF newborn screening works and encourage them to investigate symptoms of CF even if screening results are negative. "I certainly don't want people to get the idea that newborn screening is not a good thing. It's going to pick up at least 95% of patients with CF," he said. "But this paper is a cautionary note to clinicians that these are screening tests—not a diagnostic test—and there are still many different ways that newborn screening can miss a diagnosis of CF." Rock is division head of pediatric pulmonology, director of the Cystic Fibrosis Center, and director of the pediatric pulmonology fellowship program at the University of Wisconsin at Madison School of Medicine and Public Health. He also chairs the state's CF newborn screening advisory group.

The study divides the 14 trouble spots among three stages, similar to the familiar pre-analytical, analytical, and post-analytical phases of testing. First, errors can come from the newborn nursery, where specimen quality and labeling are important. Second, a majority of the study's 14 opportunities for error lie with the centralized newborn screening lab. These include mundane yet calamitous problems like the mix-up of specimens, or failure to report results to providers. Then there are the more complex issues of assay cutoffs and the rapidly changing constellation of DNA mutations. Finally, follow-up can also fail. In the study, this area covers miscommunication of newborn screen results, and errors in measuring or inappropriate cutoffs for sweat chloride.

For the central screening lab, the immunoreactive tryspinogen (IRT) assay figures prominently in the study's problem list. Babies with CF usually have high blood levels of IRT, an enzyme made in the pancreas. Both the analytical and mathematical components of the assay need improvement, according to the authors. Of particular concern in the assay's cutoff. Even a very low cutoff at the 96th percentile yields false negatives. To deal with this issue, each state also approaches the IRT cutoff differently: some have chosen various fixed cutoffs, and others use a floating cutoff. States also differ on whether they follow up with a second IRT assay or with DNA mutation testing.

In one of the illustrative cases in the study, an infant presents with CF symptoms at 11 months of age. The Wisconsin newborn screening program had barely missed her. The infant's newborn IRT screen was 66 ng/mL, but she was born on a day when the state's floating 96th percentile cutoff reached 67 ng/mL. The Wisconsin system considers the upper 4 percentile abnormal, with the cutoff determined according to each day's run.

However, there are good reasons some states use a floating cutoff, according to Rock. "Here in Wisconsin, we've found that IRT values are not consistent from one season to the other, probably because of samples exposed to cold temperatures in the winter when they're mailed to the lab in Madison. We also know that there can be significant lot-to-lot variability of IRT values," he said. "Considering this variability both by season and by reagent log, we think that the floating cutoff makes more sense for us. Other newborn screening programs have looked at this and have not seen the same kind of variability."

The assay also poses problems for IRT/IRT algorithm states due to the changing values of the enzyme according to the infant's age, the authors note. For example, in the Colorado screening program, a 2009 study found a 13% false negative rate that was attributed to repeat IRT values.

For states with an IRT/DNA algorithm, the DNA testing continues to be become more complicated, according to Rock. Every state is different in terms of which mutations have made it onto the newborn screening panel. It is estimated that approximately 190 of the close to 2,000 mutations in the CF gene cause disease, according to Rock. The American College of Medical Genetics recommends 23, yet some states only test for one common mutation. Others, like Massachusetts, may include 30 to 40 mutations. "The field of CF has become much more complicated with genetic testing," Rock said. "We've known for years that there is full-blown CF, and we've also known for years that there is non-classic or atypical CF, and now we're seeing many gradations of CF, from full blown disease to something called cystic fibrosis transmembrane conductance regulator-related dysfunction, where there may only be one isolated problem."

Especially significant for many labs, the study highlights errors in follow-up, focusing on potential problems with sweat chloride testing by quantitative pilocarpine iontophoresis. Although still considered the gold standard for diagnosing CF, sweat chloride testing faces several important problems, the authors note. In one of the illustrative cases, a 2-month-old child has a sweat test at a local hospital lab, with a sweat chloride value of 21 mmol/L, so the family is told the baby doesn't have CF. Later, the child at 16 months is referred to a CF center due to cough and weight loss. The CF center finds a sweat chloride level more than twice as high.

Because sweat testing is very complex, only results from CF Foundation (CFF)-accredited labs should be accepted, according to Rock. "Pediatric pulmonologists only trust sweat tests done in accredited CF centers," Rock said. "It's a very complicated test to run. It's all hands-on—stimulating the sweat glands, putting the filter paper or gauze on the arm, collecting the sweat, and then analyzing the specimen with a chloridometer—so if it's a not a very experienced technician that does it frequently to maintain proficiency, there are lots of places where errors can be made."

"CFF-accredited labs are definitely held to a higher standard," commented Shannon Haymond, PhD. "Sweat collection and testing are really difficult procedures in which to keep staff proficient and standardized, especially if not performed on a frequent basis. We adhere to specific requirements of the CFF that are based on CLSI guidelines and CAP regulations and are focused on accurate diagnosis and quality improvement." Haymond is the director of clinical chemistry at Children's Memorial Hospital and assistant professor of pathology at Northwestern University's Feinberg School of Medicine in Chicago, Ill. She was not associated with the study.

Although CFF recommends that all sweat tests be performed at CFF-accredited centers, Haymond noted that at least in her state, non-accredited centers are still included in state lists of locations for follow-up of abnormal newborn screens.

As a lab in a children's hospital that is also a CF referral site, Haymond's lab routinely performs sweat tests on older children in addition to newborns, despite universal screening. For this reason, she agrees with the authors of the paper that physicians should not become complacent, but continue to order sweat testing when they have suspicion of CF. "CF is a unique challenge because it has such a highly variable phenotype," Haymond said. "Sweat testing on older children that are clinically complex or acutely ill often involves difficulties for adequate collection and accurate testing but we've been successful in engaging our clinician counterparts, through partnering with our CFF director, to explain the limitations of such requests and to accommodate the need for testing while following the CFF standards."

The authors of the study exhort clinicians to remain vigilant. "Care providers, knowing that CF newborn screening is now universal in the United States, could become complacent in considering a CF diagnosis due to neonatal screening," they wrote.

Haymond emphasized that many of the potential sources of error described in the paper may apply to other newborn screening tests as well. "This is a point that I try to make when teaching or responding to calls from clinicians about newborn screening or difficult cases where the newborn screening results were 'normal'," she said. "Each state regulates its own menu, menus change over time, and there are limitations to be aware of related to specimen collection and stability, cutoffs, and the tests themselves. Newborn screening is a highly successful public health program but it should not be mistaken as comprehensive or diagnostic."