Draft Genetic Test Review
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Cystic Fibrosis
Analytic Validity
(1.14MB)
ANALYTIC VALIDITY
Question 8: Is the test qualitative or quantitative?
Question 9: How often is a test positive when a mutation is present?
Question 10: How often is the test negative when a mutation is not present?
Question 11: Is an internal QC program defined and externally monitored?
Question 12: Have repeated measurements been made on specimens?
Question 13. What is the within- and between-laboratory precision?
Question 14: If appropriate, how is confirmatory testing performed to resolve false positives in a timely manner?
Question 15: What range of patient specimens has been tested?
Question 16: How often does the test fail to give a useable result?
Question 17: How similar are results obtained in multiple laboratories using the same, or different, technology?
ANALYTIC VALIDITY
Question 13: What is the within- and between-laboratory precision?
This question is not applicable to prenatal screening for cystic fibrosis, since such testing is qualitative. This question is only relevant to quantitative measurements such as repeat sizing.
ANALYTIC VALIDITY
Question 14: If appropriate, how is confirmatory testing performed to resolve false positive results in a timely manner?
Summary
- Confirmatory testing is additional testing to confirm the finding of a mutation(s)
- Such testing should be considered when a carrier, carrier couple, or affected fetus is identified
- It is likely to be useful in selected circumstances, because of occasional false positive test results
- There is little information about how often confirmatory testing corrects an error
- The type of confirmatory testing depends on the clinical circumstances, sample type and testing methodology
- Supplementary testing might occasionally be necessary
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Definition
Confirmatory testing is performed to ensure that the initially positive test result is correct. For example, rerunning a specimen that was positive for G551D in order to ensure that it was correct is considered confirmatory testing. Reflexive testing is different from confirmatory testing in that other mutations or polymorphisms are being analyzed to aid in the interpretation of positive results. For example testing an unexpected homozygous delf508 individual for the presence of the benign polymorphism delF508C would be considered reflexive testing. This is also the case in testing for the 5T/7T/9T polymorphism after identifying the R117H mutation.
In prenatal screening for cystic fibrosis, confirmatory testing of some type should be considered in the following three circumstances:
- A cystic fibrosis mutation is identified in an individual
- A cystic fibrosis mutation is identified in both members of a couple
- A fetus with two cystic fibrosis mutations is identified
Four distinct types of confirmatory testing could be utilized, depending on the testing protocols in place and the circumstances in which the positive test result is obtained.
- Repeating the same test protocol on another aliquot of the same specimen
- Repeating the same test protocol on a different (or further processed)* specimen
- Performing a different test protocol on another aliquot of the same specimen
- Performing a different test protocol on a different (or further processed)* specimen
* further processing would include, for example, culturing fetal cells obtained via chorion villus sampling
Confirmatory testing in a prenatal cystic fibrosis screening program
In the following paragraphs, the four types of confirmatory testing are examined in each of the circumstances in which a mutation is detected. When a mutation is identified in a screened individual (about 1 in 29 non-Hispanic Caucasians), laboratories may repeat their testing protocol on another aliquot of the same sample in order to rule out sample mishandling or laboratory sample mix-up. Only in rare circumstances will any other type of confirmatory testing be performed. Rare findings, such as a previously unknown, asymptomatic homozygote might warrant confirmation with another sample, possibly with a different methodology.
In the event that a laboratory identifies a mutation in both members of a screened couple (occurring about 1 in 900 non-Hispanic Caucasian couples), further confirmatory testing might also be warranted in certain circumstances. For example, if the initial specimens were mouthwash or buccal scraping, laboratories might consider collecting and analyzing a blood specimen to demonstrate chain of custody, prior to the option of invasive testing. Some laboratories may want to verify the results using a different testing protocol, or send an aliquot of the sample to a reference laboratory for confirmation. It is important to ensure accurate results, since diagnostic testing will be an option for this couple not only for this pregnancy, but also for all subsequent pregnancies.
Some laboratories perform DNA analysis for cystic fibrosis on amniotic fluid cells prior to culture, but require confirmation on cultured cells from the same fluid. When a positive test result is identified using cells obtained from the fetus (amniotic fluid, chorion villus sampling, or umbilical blood), testing may be repeated in a second laboratory to ensure accurate results, but most experienced diagnostic laboratories feel confident in their results. There are no other tests available for confirmation in the prenatal setting; the molecular test is the definitive test. It is especially critical, in this diagnostic setting, to ensure accurate results.
Importance of confirmatory testing
The analytic specificity is currently estimated to be 97.9 percent (Question 10). It is important, therefore, to determine how often ‘false positive' results will be identified upon confirmatory testing. If the error is due to clerical or laboratory sample mix-up, simple retesting of an additional aliquot may be sufficient to identify and correct the error. Given that proficiency testing in Europe found 90 percent of the errors to be of this type (Dequeker and Cassiman, 2000), confirmatory testing can be expected to eliminate many of the false positive results. This issue is dealt with in more detail under Clinical Performance (Questions 19 and 20).
Other additional testing in a prenatal cystic fibrosis screening program
A supplementary test (reflexive testing for the 5T variant) has been recommended when the R117H mutation is identified (Grody et al., 2001). The finding of the R117H allele on a 5T background (in cis) is interpreted as a classic cystic fibrosis mutation, whereas R117H on a different background may be associated with congenital bilateral absence of the vas deferens (CBAVD) or other non-cystic fibrosis phenotypes.
The need for a second type of supplementary test arises when a delF508 allele is identified by certain types of DNA methodologies. A few analytic methods may not always distinguish between delF508 (wild type) and several benign polymorphisms (e.g., delF508C). Assaying the sample using a second test method (such as gel analysis or a method known not to cross react with these benign polymorphisms) will allow the genotype to be correctly identified and interpreted. Such testing could also be useful when an unexpected homozygous (or compound heterozygous) individual is identified.
References
Grody WW, Cutting GR, Klinger KW, Richards CS, Watson MS, Desnick RJ. 2001. Laboratory standards and guidelines for population-based cystic fibrosis carrier screening. Genet Med 3:149-154.
Cuppens H, Cassiman JJ. 1995. A quality control study of CFTR mutation screening in 40 different European laboratories. The European Concerted Action on Cystic Fibrosis. Eur J Hum Genet 3:235-245.
Dequeker E, Cassiman J. 2000. Genetic Proficiency testing in diagnostic laboratories – quality control is the message. Am J Hum Genet 67:A274
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