Available Assays

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Prior to 2001, professional recommendations limited CF carrier testing to individuals with a family history and their partners. Testing laboratories offered laboratory-developed assays using standard platforms, including reverse and forward dot blot, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)

analysis, oligonucleotide ligation assays (OLA), sequence specific primer-polymerase chain reaction (SSP-PCR), various methods for exon scanning, and sequencing. Techniques reported by 50 laboratories that participated in a proficiency testing survey from the ACMG/College of American Pathologists (CAP) in 2002 were probe hybridization (38%), OLA (26%), PCR-RFLP (18%), SSP-PCR (16%), sequencing (16%), mutation scanning (8%), and other (26%).

Currently, there are several available analyte-specific reagents (ASRs). Clinical CF tests using these ASRs are considered laboratory-developed tests and must be validated by the laboratory prior to implementation. Technical standards and guidelines for CF mutation testing have been published by the ACMG5 and are available at http://www.acmg.net. These guidelines address the technology platforms used in commercial ASRs and laboratory-developed tests for CF mutations.

The available ASRs are summarized in Table 10-2 with additional vendors anticipated to develop ASRs in the future. All are robust and include at least the ACMG/ACOG minimum core mutation panel, but vary considerably with respect to criteria that laboratories consider for platform adoption: reagent/royalty costs, footprint of instrumentation, throughput, flexibility, and data analysis. All but the Invader platform are PCR based, requiring a royalty, which varies among academic, hospital, and commercial laboratories; the PCR royalty is built into the Roche ASR. Reagent costs per patient are significant for all of the ASRs. The commercial ASRs are not likely to be submitted for FDA approval in the foreseeable future until the ACMG/ACOG mutation panel has undergone additional review.

The Nanogen and ABI platforms offer both semiauto-mated detection and data analysis. Both require capital

Table 10-2. Commercially Available Analyte Specific Reagents




Nanogen (San




Third Wave

(Indianapolis, IN)

(Ghent, Belgium)

Diego, CA)


(Indianapolis, IN)





(Madison, WI)

Oxford, UK)



PCR target

PCR target

PCR target

ARMS target

PCR target

PCR target

PCR and signal



amplification, chip





OLA, and

and reverse



and reverse


and FRET


line blot

and FRET


line blot

primer extension,



detection on


hybridization to

of alleles

an electronic

universal tags,


and sorting on

Luminex 100


Product or




Elucigene CF

CF Gold










12- and 17-

100-site chip

Slab gel


96-well plate

96-well plate





FRET, flourescence resonance energy transfer; PCR, polymerase chain reaction, ARMS, amplification refractory mutation system.

purchase of an instrument that has a relatively large footprint. The assay format for both is flexible and easily customized. Potentially, both are amenable to high throughput testing and can be used as a consolidated work platform for other assays.

The Roche and Innogenetics ASRs are reverse line blots that are in widespread use because of early availability and ease of validation in the laboratory. The Inno-Lipa detection system can be semiautomated using a customized instrument. Both are amenable to low and high test volumes but are closed platforms such that additional mutations cannot be added by the laboratory. Current versions require manual analysis of the strip data, but both companies are developing automated data-analysis software.

Both the Roche and Innogenetics probe-hybridization assays include the intron 8 5T/7T/9T variant and the exon 10 polymorphisms in the first-line assay, rather than as a reflex test as recommended by ACMG and ACOG. However, positioning of intron 8 variant probes at the bottom of strips allows strips to be cut to remove the polyT probes from first-line testing. Laboratories obtaining genotypes for the intron 8 polyT variant for all patients are required to report the results, as the technical guidelines require reporting of all patient data. This may be useful for detection of males with CBAVD, since the 5T allele is associated with this condition, but this allele is also present in 5% to 10% of the general population. Several physicians, not recognizing that this allele is benign in the absence of a second mutation, have ordered amniocentesis for couples for which both partners are mutation negative but one member of the couple carries the 5T allele.5 Because amniocentesis is not without risk, this is considered a misuse of the data.

The Orchid ASR is a manual procedure that features gel-based detection of sequence-specific primer (SSP) ampli-cons. Although the amplifications are multiplexed, samples from each patient are loaded into several gel lanes and, as such,this closed platform may be best suited for lower-test-volume laboratories.

The Tm Bioscience assay incorporates multiplex PCR and multiplex allele-specific primer extension with a proprietary Universal Tag sorting system on the Luminex 100 xMAP platform. This is a high-throughput assay that incorporates automated software analysis for genotype calls.

The Third Wave Technologies assay requires only pipetting steps and is easily automated using a liquid handler. Thus, this assay is potentially low cost, requiring only a fluorescent plate reader. Detection However, the assay is unique among CF ASRs in that it combines limited cycles of PCR and relies on signal amplification using the Invader FRET technology. Because this assay format is not multi-plexible, the detection steps are performed on a custom "InPlex" card, in which reaction products are distributed to wells of dried down reagents, each specific for the detection of a single mutant allele.

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