Draft Genetic Test Review

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Cystic Fibrosis
Analytic Validity

(1.14MB)

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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?

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ANALYTIC VALIDITY

Question 15: What range of patient specimens has been tested?

Cystic fibrosis mutation analysis has been successfully performed in a variety of specimens using available methodologies.

Screening can be performed on:

• whole blood (purified DNA and lysates),
• buccal lysates (cheekbrush, swab and mouthwash), or
• dried blood spots.

Diagnostic testing of the fetus can be performed on:

• direct and cultured amniotic fluid cells,
• chorionic villi collected via chorionic villus sampling (CVS),
• cells obtained via percutaneous umbilical blood sampling (PUBS)

Pre-implantation diagnostic testing can be carried out on a single cell.

Molecular confirmation of diagnosis can be performed on products of conception and tissue samples.

Blood samples are the most reliable method of collecting large amounts of high quality DNA, but a trained phlebotomist is needed, thereby increasing costs and requiring that specimens be collected at a medical facility. Buccal cells obtained by scraping, brushing or mouthwash yield adequate amounts of DNA for screening purposes (Doherty et al., 1996; Loader et al., 1996; Witt et al., 1996; Grody et al., 1997). This technique can be used to collect samples at the physician's office or at home. Buccal samples have the disadvantage of less DNA, higher failure rates, and less documentation of chain of custody. Buccal lysates can be frozen and stored for years and still be tested successfully (Bradley et al., 1998). Dried blood spots can also be used for PCR-based testing, and experience has been gained in newborn pilot screening trials (Summary of newborn trials contained in Question 21). However, they have not routinely been used in prenatal cystic fibrosis pilot screening programs. In an informal survey of several commercial laboratories offering cystic fibrosis testing, none accepted blood spots (Gasparini et al., 1999 (S Richards, personal communication).

For diagnostic purposes, it is most difficult to analyze fetal cells prior to culturing due to limited numbers of cells and the high potential for maternal cell contamination. When fetal specimens are tested, it is the laboratory's responsibility to assess maternal cell contamination.

References

Bradley LA, Johnson DD, Palomaki GE, Haddow JE, Robertson NH, Ferrie RM. 1998. J Med Screen 5:34-36.

Doherty RA, Palomaki GE, Kloza EM, Erickson JL, Haddow JE. 1996. Couple-based prenatal screening for cystic fibrosis in primary care settings. Prenat Diagn 16:307-404.

Gasparini P, Arbustini E, Restagno, Zelante I, Stanziale P, Gatta L, Sbaiz L, Sedita AM, Banchieri N, Sapone L, Fiorucci GC, Brinson E, Shulse E, Rappaport E, Fortina P. 1999. Analysis of 31 CFTR mutations by polymerase chain reaction/oligonucleotide ligation assay in a pilot screening of 4476 newborns for cystic fibrosis. J Med Screen 6:67-69.

Grody WW, Dunkel-Schetter C, Tatsugawa ZH, Fox MA, Fang CY, Cantor RM, Novak JM, Bass HN, Crandall BF. 1997. PCR-based screening for cystic fibrosis carrier mutations in an ethnically diverse pregnant population. Am J Hum Genet 60:935-947.

Loader S, Caldwell P, Kozyra A, Levenkron JC, Boehm CD, Kazazian HH Jr, Rowley PT. 1996. Cystic fibrosis carrier population screening in the primary care setting. Am J Hum Genet 59:234-247.

Witt DR, Schaefer C, Hallam P, Wi S, Blumberg B, Fishbach A, Holtzman J, Kornfeld S, Lee R, Nemzer L, Palmer R. 1996. Cystic fibrosis heterozygote screening in 5,161 pregnant women. Am J Hum Genet 58:823-835.

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ANALYTIC VALIDITY

Question 16: How often does the test fail to give a useable result?

Test ‘failures' in the pre-analytic phase of testing
In the pre-analytic phase, it may be determined that the sample is not suitable for testing because specific clinical criteria are not met, or because the sample is considered inadequate. While programs often monitor pre-analytic test cancellation rates as part of an overall quality assurance plan, these events are usually not considered a laboratory or methodologic ‘failure'. Table 2-9 lists criteria commonly used for deciding whether to reject a sample in the pre-analytic phase.

Table 2-9. Common Pre-analytic Criteria for Rejecting a Sample Submitted for Prenatal Cystic Fibrosis Screening

Rejection Criteria Based on Clinical Information

Gestational age too advanced (e.g., received after 20 or 21 weeks' gestation)

Testing already performed on a previous sample for this couple

Couple has a family history of cystic fibrosis (more extensive DNA testing may be indicated)

Rejection Criteria Based on Submitted Sample

Inadequate specimen quality  (e.g., hemolyzed blood, dried buccal sample or obvious contamination)

Inappropriate sample (e.g., whole blood with no anticoagulant or wrong anticoagulant)

Inadequate specimen labeling

Inappropriate handling prior to laboratory receipt  (e.g., sample too long in transit or exposed to extreme temperature)

Test failures during the analytic phase of testing
Failures of individual samples or assays occur when preset quality control standards are not met and test results are not reportable. Failures can arise for a number of reasons such as improperly processed samples, problems with component reagents, or equipment malfunction. Many assay failures within the clinical molecular genetic laboratory are due to operator error. Automation and programs to properly train laboratory personnel can avoid most of these problems. Only a few medical technology programs, however, currently provide adequate molecular components in their programs. Documentation of failures and subsequent corrective action is required by regulatory agencies such as CLIA and the College of American Pathologists. Unfortunately, failure rates and other information on assay robustness are often not published as part of pilot trials or method evaluations. Available data suggest, however, that repeating the analysis of an individual sample or assay run can often yield a satisfactory result.

A irretrievable assay failure occurs when an apparently suitable specimen is submitted and approved for testing, but the assay yields a result that is clinically uninterpretable. Failures of this type are most often related to the quality of the original sample. Procedural problems during specimen processing and DNA extraction can also be responsible. Success rates for obtaining clinically interpretable results are close to 100 percent for blood samples. Buccal samples have a somewhat lower success rate (98 percent to over 99 percent) as a result of poor sampling (inadequate number of cells), sample contamination, desiccation (exposure to extreme heat), or inadequate sensitivity of the testing methodology to account for the lower concentration and quality of the sample.

Post-analytic failures, such as incorrect or inadequately interpreted results are considered separately from analytic test failures, as part of a review of overall quality assurance in the Clinical Utility section (Question 32).

Gap in Knowledge: Overall, and method-specific failure rates
Clinical laboratories are required to document test failures as described above. For this reason, this type of information should be readily available from laboratories participating in external proficiency testing administered by the ACMG/CAP. This could be accomplished though the use of a supplemental question attached to a routine distribution or, alternatively, the data could be collected via an externally funded, independent project.