Draft Genetic Test Review

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Hereditary Hemochromatosis
Analytic Validity

(119KB)

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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 (analytic sensitivity)?
Question 10: How often is the test negative when a mutation is not present (analytic specificity)?
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?
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 17: How similar are results obtained in different laboratories?

Comparing results from different laboratories using the same or similar methodologies
The only potential source of data for evaluating differences in HFE test results from multiple laboratories using the same (or a similar) method would be derived from external proficiency testing. Method-specific comparisons are complicated, because laboratories in the same methodological category may be using different home-brew reagent components and protocols. For example, although three laboratories might be grouped under the ARMS methodology, one might use a prepared kit, a second might use commercially prepared analyte specific reagents (ASR), and the third might use in-house reagents. To help in comparing methodologies, the ACMG/CAP MGL Survey Participant Summary Reports have stratified results into broad methodological categories.

Comparing results from the same laboratory using different methodologies
Baty et al. (1998) compared the results of testing 46 samples using the ARMS test versus the restriction digestion method and found identical results. Similarly, Jackson et al. (1997) demonstrated the use of heteroduplex analysis for HFE genotyping and showed 100% concordance with results obtained by restriction digestion methods. Guttridge et al. (1998) described a method of sequence specific primers for PCR (PCR-SSP) for HFE analysis, tested 185 individuals previously typed using PCR-RFLP, and found complete agreement of results. Bernard et al. (1998) described a new method using fluorescent hybridization probes for HFE genotyping and compared it to the standard method of restriction enzyme digestion and gel electrophoresis. Of 117 patients and 56 controls tested, no discrepancies were noted. SSCP and capillary electrophoresis were also used to perform HFE testing on 85 patients with liver disease, and RFLP analysis was used to confirm the results (Bosserhoff et al., 1999; Wenz et al., 1999).

Neoh et al. (1999) reported a method based on fluorescence resonance energy transfer (FRET) and real time polymerase chain reaction (PCR) to identify HFE genotypes in 112 individuals. The results were compared to restriction digestion of PCR products. Agreement was found in 244 of 246 samples tested. Sequence analysis determined that the FRET analysis result was correct. Parks et al. (2001) reported a similar study in which 450 patients were tested for HFE (C282Y) using FRET analysis. Their results were compared with standard PCR and RFLP analysis, with 100% concordance. Steffensen et al. (1998) tested 200 Danish individuals for the C282Y and H63D mutations, using a sequence-specific primer method for PCR analysis (PCR-SSP) and compared their results to a standard method of PCR-RFLP with complete agreement of methods for analysis.

Other methods for testing for HFE include dHPLC analysis, with or without single-base extension (Devaney et al., 2001; Liang et al., 2001), Lightcycler (Kyger et al., 1998), and the DNA capillary array electrophoresis chips (Woolley et al., 1997), although less information is available regarding assay validation studies.

Comparing results from different laboratories using different methodologies
As part of the 2000 ACMG/CAP Molecular Genetics Laboratory external proficiency testing survey, laboratories were queried about their methodology for performing HFE mutation analysis (Table 2-15, Appendix 2). Overall, the reported methodologies were used to detect one or two mutations (with the majority of laboratories testing both C282Y and H63D). During the four years of proficiency testing (1998 through 2001) there was a high level of agreement between laboratories for detecting mutations that were targeted by their specific method, no matter which method was being used.

Gap in Knowledge: Comparison of Methods for HFE Mutation Detection In order to compare analytic validity for various testing methodologies, proficiency testing data from ACMG/CAP have been stratified by methodological category. It would also be useful to identify subsets using the same commercially available reagents (e.g., in-house reagents versus ASR). Alternatively, a previously described method for validation (Question 9 – Optimal Sources of Data) could be employed that would provide not only analytic performance for a methodology, but also comparative data between methodologies.

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Appendix 2. Analytic Methodologies used for HFE Mutation Analysis

Introduction
Table 2-15 lists categories of methodologies that are used to detect HFE mutations by laboratories participating in proficiency testing programs in the United States (ACMG/CAP MGL Survey), along with the proportions using each method. Because many laboratories utilize “home brew” assays, these categories are not necessarily homogeneous. Some methodologies are relatively labor intensive, making them more suitable for research than diagnostic laboratories. When large numbers of specimens must be tested with short turn-around times, other methodologies are needed. Commercial suppliers can provide “kits” to European or Canadian-based clinical laboratories. U.S. laboratories are not allowed to use “kits” for clinical testing but may use analyte specific reagents (ASR).

Table 2-15. Testing Methods Utilized by 90 U.S. Laboratories According to the 2002 ACMG/CAP MGL External Proficiency Testing Survey

Testing Method	Proportion of Laboratories (%)
Electrophoresis for RFLP and size analysis	64
Allele Specific Oligonucleotide (ASO)	11
Allele-specific PCR/ARMS	6
LightCycler	8
Sequencing	3
Other/Not specified	8
Total	100

 

HFE testing methodologies for screening ought to include the following characteristics:

• ability to selectively identify only the C282Y mutation
• a low to moderate level of technical expertise
• a short turn-around time (one or two days)
• a high throughput (ideally, on an automated platform)
• a relatively low cost

These requirements might appear ambitious, but the evolution of other tests now used for screening in the clinical laboratory shows that these goals are achievable. For example, immunoassays that are now routinely performed were originally developed in the 1960s by investigators with in-depth knowledge of immunochemistry and radiation detection methods. Over the ensuing years, these assays were revised and streamlined by manufacturers to meet the needs of clinical laboratories, including the development of automated immunoassay systems that minimize the chance for error. For FDA approved kits, the responsibility for ensuring reagent quality and instrument performance now rests primarily with the manufacturer. The laboratory's responsibility is to monitor the quality control measures set by the manufacturer to verify that assay performance meets specifications. A further development is a computer link to the instrument that automatically transfers test results to a patient record system for reporting. Automation is more expensive than manual assays in terms of reagents and instrument rental or purchase, but the overall cost per test can be the same or lower because of the reduced labor costs. This same development is beginning to occur for HHC screening. Several commercially prepared reagents have emerged, and their attributes are summarized in Table 2-16.

[Table 2-16 and the following notes are still under construction]

Table 2-16. Characteristics of Commercial Analyte Specific Reagents (ASR) to Detect HFE Mutation

	Commercial HFE Mutation Detection System
	Bio-Rad	Nanogen	LightCycler	Orchid
Characteristic
1. Method Type
2. Company
3. Mutations
4. Robustness
5. Special equipment
6. Total time (days)
7. Cost per patient
8. Advantages
9. Disadvantages
For more information

Notes pertaining to Table 2-16:

1. Method type: Methods displayed are those that are most commonly used and that are suitable for large-scale hemochromatosis screening. These include the allele specific oligonucleotide assay (ASO). The ASO assay uses reverse ASO technology, with the oligonucleotides bound to the microplate surface. Biotinylated DNA is bound to the ASOs; Streptavidin horseradish peroxidase is used bound to the biotin, oxidizes a substrate, and results in a colorimetric change. Detection is done by colorimetric analysis using a plate reader. Two wells are required for each allele analyzed. Genotype is determined by a ratio of absorbance. An alternate means of amplification of DNA is available in this ASR format and is termed LLA or linked linear amplification (Linked Linear Amplification: A New Method for Amplification of DNA. Clin Chem 47:31-40 (2001). Both standard PCR and LLA ASRs are available. This assay has been validated by the manufacturer against other molecular methods for performing hemochromatosis testing. Whole blood was the only sample type that was tested, according to the manufacturer. For more information about methodologies, including a description and set of references, see www.bio-rad.com.
2. Company: Commercial reagents have not been approved by the FDA.  However, reagents have had one level of QC/QA performed by the manufacturer, as specified by the FDA's Analyte Specific Reagent (ASR) rule.  ASRs are produced using GMP and have undergone rigorous quality control testing in house.  As Bio-Rad owns the patent for hereditary hemochromatosis, there are no other commercially available manufactured reagents for this test.
3. Mutation(s):  This is the hereditary hemochromatosis mutation(s) that can be detected by the testing protocol.  This ASR is currently designed to test for both C282Y and H63D.  Currently, a laboratory that uses this reagent must test for both alleles.  The S65C mutation may be added to the ASR.  Initially there was some concern of interference with ASO binding in H63D/S65C compound heterozygotes, as the ASOs were not designed to distinguish these two alleles.  Thus, an H63D/S65C compound heterozygote could appear as an H63D homozygote.  The current ASR does not include the S65C mutation.
4. Robustness: Robustness describes how consistently and reliably a set of reagents performs when used by different laboratories, under varying conditions, and on different sample types (e.g., blood, buccal smears).
5. Special equipment: Some manufacturers require that specialized equipment be used to perform their assays.  Although initially more costly, the equipment may allow more samples to be tested.  The Bio-Rad ASRs require a plate reader for the colorimetric analysis detection.  Currently, there is no specific software associated with the interpretation of genotype, and laboratories are left to design their own system.  Some use Excel spreadsheets.  A more automated system with the flexibility to set cutoffs would be desirable.
6. Total time: Estimated time to complete assay, including sample processing and reporting.  This method only requires one day, but laboratories may choose to extend the process to a second day for more convenient scheduling.
7. Cost per patient: Costs for the reagents and licenses to perform hereditary hemochromatosis testing are extremely variable.  Some laboratories perform ‘in-house' assays with relatively low reagent costs.  In such cases, the cost of technical time for reagent preparation and QC/QA must also be considered.  Costs of analyte specific reagents (ASR) can be relatively high, compared to traditional biochemical assays.  However, the savings in technical staff time for preparation and QC/QA can offset reagent costs.  For screening, the relevant figure is the cost per patient tested, rather than the cost per mutation tested.
8. Advantages: Reagents for hereditary hemochromatosis screening should have high throughput with relatively low labor costs.  Assays that can be efficiently automated can be cost effective.  Peer-reviewed analytic validity data are helpful for validation.

Newer testing technology platforms with high potential for hereditary hemochromatosis testing include various hybridization strategies (Roche and Luminex), arrayed primer extension (Orchid), mass spectrometry (Sequenom), and sequence analysis (Pyrosequencing).  However, there are no existing data that accurately compare these technologies with currently utilized methodologies or with each other.

References

Baty D, Terron Kwiatkowski A, Mechan D, Harris A, Pippard MJ, Goudie D.  1998.  Development of a multiplex ARMS test for mutations in the HFE gene associated with hereditary hemochromatosis.  J Clin Pathol  51:73-74.

Bernard PS, Ajioka RS, Kushner JP, Wittwer CT.  1998.  Homogeneous multiplex genotyping of hemochromatosis mutations with fluorescent hybridization probes.  Am J Pathol  153:1055-1061.

Bosserhoff AK, Seegers S, Hellerbrand C, Scholmerich J, Buttner R.  1999.  Rapid genetic screening for hemochromatosis using automated SSCP-based capillary electrophoresis (SSCP-CE).  Biotechniques  26:1106-1110.

Devaney JM, Pettit EL, Kaler SG, Vallone PM, Butler JM, Marino MA.  2001.  Genotyping of two mutations in the HFE gene using single-base extension and high-performance liquid chromatography.  Anal Chem  73:620-624.

Guttridge MG, Thompson J, Worwood M, Darke C.  1998.  Rapid detection of genetic mutations associated with haemochromatosis.  Vox Sang  75:253-256.

Jackson HA, Bowen DJ, Worwood M.  1997.  Rapid genetic screening for haemochromatosis using heteroduplex technology.  Br J Haematol  98:856-859.

Kyger EM, Krevolin MD, Powell MJ.  1998.  Detection of the hereditary hemochromatosis gene mutation by real-time fluorescence polymerase chain reaction and peptide nucleic acid clamping.  Anal Biochem  260:142-148.

Liang Q, Davis PA, Thompson BH, Simpson JT.  2001.  High-performance liquid chromatography multiplex detection of two single nucleotide mutations associated with hereditary hemochromatosis.  J Chromatogr B Biomed Sci Appl  754:265-270.

Neoh SH, Brisco MJ, Firgaira FA, Trainor KJ, Turner DR, Morley AA.  1999.  Rapid detection of the factor V Leiden (1691G>A) and haemochromatosis (845G>A) mutation by fluorescence resonance energy transfer (FRET) and real time PCR.  J Clin Pathol  52:766-769.

Parks SB, Popovich BW, Press RD.  2001.  Real-time polymerase chain reaction with fluorescent hybridization probes for the detection of prevalent mutations causing common thrombophilic and iron overload phenotypes.  Am J Clin Pathol  115:439-447.

Reyes AA, Ugozzoli LA, Lowery JD, Breneman JW 3^rd, Hixson CS, Press RD, et al.  2001.  Linked linear amplification: a new method for the amplification of DNA.  Clin Chem  47:31-40.

Steffensen R, Varming K, Jersild C.  1998.  Determination of gene frequencies for two common haemochromatosis mutations in the Danish population by a novel polymerase chain reaction with sequence-specific primers.  Tissue Antigens  52: 230-235.

Wenz HM, Baumhueter S, Ramachandra S, Worwood M.  1999.  A rapid automated SSCP multiplex capillary electrophoresis protocol that detects two common mutations implicated in hereditary hemochromatosis (HH).  Hum Genet  104: 29-35.

Woolley AT, Sensabaugh GF, Mathies RA.  1997.  High-speed DNA genotyping using microfabricated capillary array electrophoresis chips.  Anal Chem  69:2181-2186.