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Journal of Molecular Diagnostics, Vol. 12, No. 5, September 2010 Copyright © American Society for Investigative Pathology and the Association for Molecular Pathology DOI: 10.2353/jmoldx.2010.090233

The Suitability of Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry in a Laboratory Developed Test Using Cystic Fibrosis Carrier Screening as a Model

Daniel H. Farkas,* Nicholas E. Miltgen,* Jay Stoerker,† Dirk van den Boom,‡ W. Edward Highsmith,§ Lesley Cagasan,¶ Ron McCullough,¶ Reinhold Mueller,¶ Lin Tang,¶ John Tynan,¶ Courtney Tate,储 and Allan Bombard‡ From the Sequenom Center for Molecular Medicine,* Grand Rapids, Michigan; the Sequenom Center for Molecular Medicine,† Ann Arbor, Michigan; Sequenom, Inc.,‡ San Diego, California; the Mayo Clinic,§ Rochester, Minnesota; Sequenom Center for Molecular Medicine,¶ San Diego, California; and Osmetech, Inc.,储 Pasadena, California

We designed a laboratory developed test (LDT) by using an open platform for mutation/polymorphism detection. Using a 108-member (mutation plus variant) cystic fibrosis carrier screening panel as a model, we completed the last phase of LDT validation by using matrix-assisted laser desorption/ionization time of flight mass spectrometry. Panel customization was accomplished via specific amplification primer and extension probe design. Amplified genomic DNA was subjected to allele specific , single base extension endpoint analysis by mass spectrometry for inspection of the cystic fibrosis transmembrane regulator gene (NM_000492.3). The panel of mutations and variants was tested against 386 blinded samples supplied by “authority” laboratories highly experienced in cystic fibrosis transmembrane regulator genotyping; >98% concordance was observed. All discrepant and discordant results were resolved satisfactorily. Taken together , these results describe the concluding portion of the LDT validation process and the use of mass spectrometry to detect a large number of complex reactions within a single run as well as its suitability as a platform appropriate for interrogation of scores to hundreds of targets. (J Mol Diagn 2010, 12:611– 619;

Southern blot-based detection of immunoglobulin gene rearrangements in B cell leukemias and lymphomas,2 examination of expansions of the trinucleotide repeat associated with disease,3,4 and amplification-based detection of the cryptic plasmid in Chlamydia trachomatis.5 What these examples have in common, as well as the majority of tests listed on the Association for Molecular Pathology website under “FDA Cleared/Approved Molecular Diagnostics Tests,” is their target number: one. Over time, the list began to expand to include multiplex panels, from the smallest multiplex target panel possible, two (C. trachomatis/Neisseria gonorrhoeae), to sequencing-based detection of multiple mutations within the HIV-1 genome that contribute to antiviral resistance, to panels ranging from 23 to many dozens of cystic fibrosis (CF)-causing mutations, to the more complex test type called in vitro diagnostic multivariate index assay in which, for example, scores of genes are interrogated to predict breast cancer recurrence or 1500 gene expression profiles are examined to determine tumor tissue of origin. This progression from tests interrogating one target to tests interrogating dozens, scores, even hundreds of targets has been both sensible and inevitable. With the completion of the sequencing of the human genome and the maturation of molecular diagnostics, it is not surprising that complex diseases have been associated with complex, ie, multigenic or multiple targets within a gene, pathogenesis. One finds under 100 tests when one examines the above named list of Food and Drug Administration-cleared and approved tests. Molecular diagnosticians have supplemented well this list of tests with laboratory developed tests (LDTs)6 that are widely used in the community. Indeed, the prevalence and indispensability of LDTs led to a College of American Pathologists

Accepted for publication April 21, 2010.

DOI: 10.2353/jmoldx.2010.090233)

D.H.F., N.E.M., J.S., D.v.d.B., L.C., R.M., R.M., L.T., J.T., and A.B. are employed by Sequenom or the Sequenom Center for Molecular Medicine. None of the other authors declare any relevant financial relationships.

The use of DNA- and RNA-based testing in laboratory medicine has grown steadily and dramatically over the past 2.5 decades. Early testing examples include restriction fragment length polymorphism analysis of mutations in the ␤– globin gene causative of sickle cell anemia,1

Supplemental material for this article can be found on http://jmd. amjpathol.org. Address reprint requests to Daniel H. Farkas, Ph.D., Sequenom Center for Molecular Medicine, 301 Michigan St. NE, Grand Rapids, MI 49503. E-mail: [email protected].

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612 Farkas et al JMD September 2010, Vol. 12, No. 5

(CAP)-sponsored publication on principles and practices for validating an LDT.7 Molecular diagnosticians require “open” platforms to create more LDTs, particularly ones that must be designed to detect more than the small handful of targets readily interrogated by the current “workhorse” of the molecular diagnostics laboratory, real-time polymerase chain reaction-based analysis. Many of the tests on the “AMP/FDA” list are on “closed” platforms, ones where further development is not possible, or that are not suitable for investigation of a medium or high density of targets. Open platforms, on the other hand, are by definition ones that are amenable to the creation of newly developed tests for laboratory diagnostics and thus can undergo those elements of design control mandated by credentialing bodies like CAP, eg, the “Assay Validation” section of the CAP Laboratory Accreditation Inspection Checklist and as further described.7 Although DNA array technology has evolved to the point of making interrogation of thousands of targets (or more) technically feasible, most existing commercially available array-based solutions do not easily lend themselves to implementation of an LDT. Certainly, examples of LDTs with scores to hundreds of targets exist.8 –11 When the target specificity is incorporated into the “predetection” phase of an assay, a multiplex detection platform dedicated only to differentiation of those targets lends itself well to customization, as described here and exemplified.10 We used an open detection platform, matrix-assisted, laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry, in an LDT for cystic fibrosis transmembrane regulator (CFTR) mutation screening via the development of a 108-member (mutations plus variants) panel as a model. Detection of the targets in this panel is based on multiplex PCR and assay- (mutation- or variant-) specific single base extension (SBE) reactions to differentiate normal from mutant or variant alleles. Detection of the resultant extension products using MALDI-TOF mass spectrometry was accomplished by simple visual inspection of well-resolved mass spectra that differentiate normal from mutant (or variant) products based on the unique molecular weight of the product containing the nucleotide representing one allele or the other. The CFTR screening mutation panel was customized not within the detection array but rather in the biochemistry of test design (multiplex PCR and SBE assays). There is controversy over the appropriateness of CFTR mutation panels with more than the standard 23 mutations recommended by the American College of Medical Genetics (ACMG) and the American College of Obstetricians and Gynecologists.12 At the same time, not only are there Food and Drug Administration-cleared “kits” on the market that may be used to detect ⬎23 mutations, but also, there are plausible reasons for enhanced numbers beyond the standard 23.13,14 We do not debate these points here but rather stress that 108 CFTR targets were chosen to demonstrate the suitability of the MALDI-TOF mass spectrometry platform for customization of detection of medium density panels. Improved CF coverage

rates were calculated by using Bayseian analysis and described below. Because of the increasing difficulty in assigning a single ethnicity to individual Americans, expanded CF mutation panels may provide increased detection rates for many individuals. For example, it has been reported that approximately 6% of CF carriers would be missed by using the standard 23-mutation CF carrier screen (listed in Table 1).14 We report (1) the concluding step of a design control process15 for implementation of an LDT, specifically, the last phase of analytical validation, as recently proposed by the CAP,7 stressing here and below that this report is not meant as a model for or description of full analytical validation; (2) a 108-member (mutations plus variants) panel for CF screening; and (3) the suitability and flexibility of mass spectrometry as a platform for multiplex target detection and clinical laboratory developed tests.

Materials and Methods Specimens We do not describe in this report the full validation process.7 Rather, to complete the generation of validation data by using an LDT that had been optimized and verified (data not shown) for detection of 108 CFTR mutations and variants of the gene (Table 1), a cohort of 12 blood specimens (genomic DNA was isolated by using Qiagen EZ1 DNA Blood 350 ␮l Kits; Qiagen Inc., Valencia, CA) and 374 purified genomic DNAs was assembled, obtained from nine “authority” laboratories (Table 2) experienced in CFTR genotyping. DNAs, both positive and negative for multiple CFTR mutations interrogated by those laboratories, were represented. All authority laboratories provided “blinded” specimens, ie, genotype results were unknown to the testing laboratory. Once experimental results were obtained, sealed results provided by three of the laboratories were unsealed by a neutral third party medical professional. This independent professional compared the authority laboratory results with our laboratory test results. The other six authority laboratories received our laboratory test results and reported back on concordance, discordance, or discrepancies.

Controls Controls for all assays (normal/mutant/variant) were included; plasmid-based cloned genomic DNAs or synthetic oligonucleotides were obtained from two manufacturers. Cystic fibrosis Panel III (Maine Molecular Quality Controls Inc., Scarborough, ME) contains two plasmids that cover 76 of the 108 mutations and variants in the panel as two equimolar alleles (normal and mutant) mimicking heterozygous genotypes. The remaining 32 assays were controlled by specific oligonucleotides manufactured by Bioneer (Alameda, CA), which appear as homozygous mutant genotypes. Herring sperm DNA (Invitrogen Inc., Carlsbad, CA) was used as a negative amplification control.

Mass Spectrometry for CF Carrier Screening 613 JMD September 2010, Vol. 12, No. 5

Table 1. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61

CFTR Laboratory Developed Test Mutations and Variants List and the American College of Obstetricians and Gynecologists and the ACMG 23 Mutation Screening Panel c.928_937delTTC (⌬F311) c.1521_1523delCTT (⌬F508)* c.1519_1521delATC (⌬I507)* c.948delT (1078delT) c.1156insTA (1288insTA) c.1545delTA (1677delTA) c.1585 ⫺ 1G⬎A (1717 ⫺ 1G⬎A)* c.1680 ⫺ 1G⬎A (1812 ⫺ 1G⬎A) c.1766 ⫹ 1G⬎A (1898 ⫹ 1G⬎A)* c.1776 ⫹ 1G⬎T (1898 ⫹ 1G⬎T c.1779 ⫹ 1G⬎C (1898 ⫹ 1G⬎C) c.1779 ⫹ 5G⬎T (1898 ⫹ 5G⬎T) c.1817del84 (1949del84) c.1911delG (2043delG) c.1923_1932delCTCAAAACTinsA (2055del9⬎A) c.1976delA (2108delA) c.2011delT (2143delT) c.2047AA⬎G (2183delAA⬎G) c.2052delA (2184delA)* c.2052insA (2184insA) c.2175insA (2307insA) c.2657 ⫹ 5G⬎A (2789 ⫹ 5G⬎A)* c.2737insG (2869insG) c.165 ⫺ 1T⬎A (296(⫹2)T⬎A) c.2988 ⫹ 1G⬎T (3120 ⫹ 1G⬎A)* c.3039delC (3171delC) c.3067_3073del6 (3199del6) c.3437delC (3659delC)* c.3535_3541del4 (3667del4) c.3659delC (3791delC) c.3718 ⫺ 2477C⬎T (3849 ⫹ 10kbC⬎T)* c.3744delA (3876delA) c.3773_3774insT (3905insT) c.262delTT (394delTT) c.3884insT (4016insT) c.273 ⫹ 1G⬎A (405 ⫹ 1G⬎A) c.273 ⫺ 3A⬎C (405 ⫹ 3A⬎C) c.274 ⫺ 1G⬎A (406 ⫺ 1G⬎A) c.312delA (444delA) c.325_327delTATinsG (457TAT⬎G) c.415delA (574delA) c.489 ⫹ 1G⬎T (621 ⫹ 1G⬎T)* c.501delT (663delT) c.579 ⫹ 1G⬎T (711 ⫹ 1G⬎T)* c.579 ⫹ 5G⬎T (711 ⫹ 5G⬎A) c.580 ⫺ 1G⬎T (712 ⫺ 1G⬎T) c.720_742del22 (852del22)* c.803delA (935delA) c.804delTA (936delTA) c.1364C⬎A (A455E)* c.1807G⬎A (A559T) c.1704C⬎A (C524X) c.165 ⫺ 5944_273 ⫹ 10251 (CFTRdele2,3) c.3459G⬎C (D1152H) c.178G⬎T (E60X) c.178G⬎T (E92X) c.532G⬎C (G178R) c.1120G⬎T (G330X) c.1507G⬎T (G480C) c.1624G⬎T (G542X)* c.1652G⬎A (G551D)*

62 63 64 64 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103

c.122G⬎A (G85E)* c.595C⬎T (H199Y) c.2128C⬎T (K710X) c.617T⬎G (L206W) c.2991T⬎C (L997F) c.3302T⬎G (M1101K) c.3302T⬎A (M1101R) c.3909C⬎G (N1303K)* c.613C⬎T (P205S) c.1721C⬎A (P574H)) c.3713A⬎G (Q1238X) c.1075C⬎A (Q359K/T360K) c.1477C⬎T (Q493X) c.1654C⬎T (Q552X) c.2668C⬎T (Q890X) c.3196C⬎T (R1066C) c.3472C⬎T (R1158X) c.3484C⬎T (R1162X)* c.349C⬎T (R117C) c.350G⬎A (R117H)* c.1000C⬎T (R334W)* c.1040G⬎A (R347H) c.1040G⬎C (R347P)* c.1055G⬎A (R352Q) c.1657C⬎T (R553X)* c.1679G⬎C (R560T)* c.2125C⬎T (R709X) c.223C⬎T (R75X) c.2290C⬎T (R764X) c.1064C⬎G (S1196X) c.3752G⬎A (S1251N) c.3764C⬎A (S1255X) c.1397C⬎A (S466X) c.1647G⬎A (S549N) c.1646A⬎C (S549R) c.1162AC⬎TA (T338I) c.3266G⬎A (W1089X) c.3612G⬎A (W1204X) c.3846G⬎A (W1282X)* c.3277C⬎A (Y1092X关C⬎A兴) c.3722C⬎G (Y1092X关C⬎G兴) c.366G⬎A (Y122X) Variants c.1210 ⫺ 12T(5_9)/c.1210 ⫺ 12关5兴/ c.1210 ⫺ 12T关9兴 (5/7/9)T c.1523T⬎G (F508C) c.1516A⬎G (I506T) c.1516A⬎G (I506V) c.1519_1521delATC (I507V)

Note: All cDNA sequence change nomenclature is based on the GenBank cDNA reference sequence NM_000492.3. *Twenty-three CFTR mutations recommended for testing by the American College of Medical Genetics.

Multiplex PCR One hundred five primer pairs, each ordered as an analyte specific reagent (Integrated DNA Technologies, Coralville, IA) component of the LDT, were used in eight

multiplex polymerase chain reactions per patient to amplify portions of the CFTR gene (NM_000492.3) containing the 108 targets (Table 1) of the test. PCR was performed in 5-␮l reaction volumes and consisted of 200 ␮mol/L dNTPs (dATP, dGTP, dCTP, dUTP), 0.25 U uracil-


Table 2.

Definitions

Authority laboratory: any of a number of clinical laboratories experienced in performing CFTR genotyping that shared blinded specimens with the developer of the LDT described in this study. Concordant: harmonious, agreeing; specifically, if the genotype “call” (assignment, post laboratory analysis) made by the LDT developer is in perfect agreement relative to the call of the “authority laboratory,” the call is concordant. Discordant: being at variance; specifically, if the genotype “call” (assignment, post laboratory analysis) made by the LDT developer did not match the call of the authority laboratory because that particular mutation was either (1) on the “authority laboratory” CFTR mutation interrogation panel but not on the developing laboratory’s panel or (2) on the developing laboratory’s CFTR mutation interrogation panel but not the “authority laboratory” panel, then that call is discordant, not erroneous or discrepant. Discrepant: differing; disagreeing; inconsistent; specifically, if the genotype “call” (assignment, post laboratory analysis) made by the developing laboratory is incorrect relative to the call of the authority laboratory, the call is discrepant. Resolution: the process whereby variance between the LDT developer’s and the authority laboratory’s results are determined thus explaining (resolving) and proving the correct result.

N-glycosylase, 1 U of FastStart TaqDNA polymerase (all preceding reagents: Roche Inc., Indianapolis, IN), and 4.125 mmol/L MgCl2 (Promega, Inc., Madison, WI), 50 ng genomic DNA, and 0.1 ␮mol/L primers. During optimization of the LDT, the individual assays for S1251N (c.3752G⬎A), 3171 delC (c.3039delC), 3120 ⫹ 1G⬎T (c.2988 ⫹ 1G⬎T), P574H (c.1721C⬎A), 621 ⫹ 1G⬎T (c.489 ⫹ 1G⬎T), D1152H (c.3459G⬎C), and 444delA (c.312delA) demonstrated insufficient amplification for successful downstream probe interrogation in their respective multiplex detection reactions. Asymmetric PCR was therefore used to amplify preferentially these targets’ reverse strands for the individual assay’s probes to bind. The reverse primer concentrations were increased as follows: P574H (c.1721C⬎A), 621 ⫹ 1G⬎T (c.489 ⫹ 1G⬎T), D1152H (c.3459G⬎C), and 444delA (c.312delA) reverse primers were increased twofold to 0.2␮mol/L; S1251N (c.3752G⬎A), 3171delC (c.3039delC), and 3120 ⫹ 1G⬎T (c.2988 ⫹ 1G⬎T) reverse primers were increased fivefold to 0.5␮mol/L. Thermal cycling conditions in a GeneAmp PCR system 9700 instrument (Applied Biosystems, Foster City, CA) were 30°C for 10 minutes, 94°C for 15 minutes followed by 45 cycles of 94°C for 20 seconds, 56°C for 30 seconds, and 72°C for 60 seconds, and an additional extension step at 72°C for 3 minutes.

Shrimp Alkaline Phosphatase Treatment Residual dNTPs from polymerase chain reactions were dephosphorylated by shrimp alkaline phosphatase (SAP) treatment by adding 0.51 U SAP (USB Corporation, Cleveland, OH) to the completed 5-␮l amplification reactions and incubated at 37°C for 20 minutes followed by a 5-minute incubation at 85°C. In the absence of SAP treat-

ment residual dNTPs are available to participate in and thus interfere with subsequent base extension reactions.

SBE Reaction Design All primers and multiplex combinations were designed by using MassARRAY System Assay Design Software version 3.1 (Sequenom, Inc., San Diego, CA). Primers, as analyte specific reagents, were obtained commercially (Integrated DNA Technologies). Due to the nature of several mutations and variants in the panel, two chemistry techniques were used in the base extension reaction. The first interrogates genotype based on SBE, using polymerase, a mixture of terminating NTPs, and an extension primer used to examine the PCR amplicon directly adjacent to the mutation site. During the extension step, the polymerase adds the complementary terminating NTP to the hybridizing extension primer/probe at the locus of interest corresponding to either the normal or mutant allele. The second extension chemistry used is called homogeneous MassEXTEND (hME) genotyping (multiple base extension), which differs from SBE chemistry in that its NTPs consist of both dideoxy- and ordinary dNTPs. In both cases (SBE and hME), the addition of the terminating base(s) to the extension primer creates a specific product with molecular weights resolvable by mass spectrometry, which can be used to assign a genotype, normal or mutant. See Supplemental Table 1 at http://jmd. amjpathol.org for extension primer sequences and their expected products.

SBE Reaction Base extension of amplified DNA targets was performed in 9-␮l reactions consisting of SAP-treated PCR products, 5 to 30 ␮mol/L extension primer, 184 ␮mol/L of each terminating NTP, SAP buffer (USB Corporation), and 3.843 U of ThermoSequenase DNA polymerase (GE Health care, Piscataway, NJ). Thermal cycling for all eight multiplexes was performed in a GeneAmp PCR system 9700 instrument: initial denaturation at 94°C for 30 seconds, 40 cycles of nested cycling at 94°C for 5 seconds, 52°C for 30 seconds and 80°C for 5 seconds with the 52°C and 80°C steps nested for 5 cycles, followed by 72°C for 3 minutes.

Preparation of Sample for Mass Spectrometry Extension reaction products were diluted with 16 ␮l molecular biology grade water and incubated with 6 mg of ammoniated clean resin (Bio-Rad, Hercules, CA) for 10 minutes with rotation to reduce salt adducts. Salt adducts generate spectral peaks at their corresponding molecular weights that may be observed after mass spectrometry and potentially interfere with analysis. Approximately 20 nL of sample were dispensed to a matrix pad of a SpectroCHIP II (Sequenom, Inc.) using a RS-1000 Nanodispenser (Sequenom, Inc.). Mass spectra were generated on a MALDI-TOF mass spectrometer (Sequenom,


Figure 1. Mass spectra of ⌬F508 (DF508) assay representative of SBE/iPlex chemistry. A: “No amplification” control (herring sperm DNA) shows no base extension of the extension primer (mass ⬇ 6079). B: Validation sample (GGC-068) genotyped as normal for the ⌬F508 assay; only the base, “A,” corresponding to the normal allele, was added resulting in the peak labeled wild-type (WT; mass ⬇ 6375); note the absence of any extension product at the mass labeled with the mutant (MUT) dotted line. C: Validation sample (GGC-072) genotyped as heterozygous for ⌬F508 as both the WT base, “A,” and mutant base, “T,” were added to the extension primer. D: Validation sample (WU-026) genotyped as homozygous mutant for ⌬F508 as only the mutant base “T” was added to the extension primer. Peaks in the spectra above (asterisks) are the extension primer of another assay in the “plex” that coincidentally is in the same mass “window.” X axis, m/z in Da; y axis, relative intensity (software calculates values from raw data). UEP, unextended primer.

Inc.) detecting positive ions of the nucleic acids in linear mode after laser desorption with a nitrogen laser (337 nm).

Data Analysis Raw data were obtained from the mass spectrometer by SpectraAQUIRE software (Sequenom, Inc.) for each sample pad and consolidated by MassARRAY System Typer version 4.0 software (Sequenom, Inc.) for visual review. Genotype calls for all 386 samples were manually assigned, independently, by two medical technologists for each assay based on measurement of the mass of the extension product, which was unique for normal and mutant alleles. Quality and ultimate acceptance of the data as supportive of a genotype “call” were based on three objective criteria: signal to noise ratio, percent conversion of extension primer to product, and peak probability (derived by Typer software). Mass spectra of the ⌬F508 assay are shown in Figure 1, A–D, and are representative of SBE chemistry. Mass spectra of the 3905insT assay are shown in Figure 2, A and B, and are representative of hME chemistry. Figure 3,

A–D, shows mass spectra that identify the deletion of CFTR exons two and three.16 Assays CFTRdele2_3, CFTR_WT_2, and CFTR_WT_3 are assessed in combination to identify and determine the zygosity of this mutation (see figure legend for explanation).

Results A comparison of CFTR genotyping results is shown in Table 3. Three hundred eighty-six specimens previously genotyped by using specific mutation panels for CFTR and obtained from nine authority labs were genotyped by the mass spectrometry procedure described above. The genotypes of these specimens were unknown to our laboratory and results were compared only after generation and documentation of genotypes using the LDT. Of these 386 specimens, 357 (92.5%) demonstrated concordant results, ie, genotype call of the authority laboratory and our laboratory were in agreement. Of these 357, 130 (36.4%) were negative for all mutations in both laboratories’ panels. Twenty-four of the 386 specimens (6.2%)

Figure 2. Mass spectra of 3905insT assay representative of hME chemistry. A: Validation sample (GGC-033) genotyped as normal for 3905insT; only the wild-type (WT) bases “G and C” were added. B: Validation sample (WU-085) genotyped as heterozygous for 3905insT; WT bases “G” and “C” and mutant bases “A, G, and C” were added to the extension primer. Peak in the spectra (asterisk) in A is the extension primer of another assay in the “plex” that coincidentally is in the same mass “window.” X axis, mass, in Da; y axis, intensity in arbitrary units.


Figure 3. The three assays (CFTRdele2_3, CFTR_WT_2, and CFTR_WT_3) are used to identify the deletion of exons two and three of the CFTR gene and to determine the zygosity of this deletion. A mass spectrum of a patient (WU-045) without the CFTR deletion on either allele is shown in A. The PCR primers for the CFTRdele2_3 assay are designed such that amplification will not occur in the presence of the two exons (A). In the absence of the deletion, the primers used for its amplification cannot anneal because their complementary target is absent.14 Therefore, in a truly normal patient, no base extension can occur. An example of a specimen (NCH-045) with the deletion of exons two and three on at least one allele is shown in B. To determine the zygosity of this specimen, the mass spectra for assays CFTR_WT_E2 and CFTR_WT_E3 must be analyzed to confirm the presence of these exons on the other allele. This specimen (NCH-045) clearly showed base extension of these other assays C and D, making the genotype heterozygous for the deletion detected in B. Had there been no base extension for NCH-045 in spectra C and D, this specimen would be homozygous for the deletion observed in B. Peaks in the spectra in A and B (asterisks) is the extension primer of another assay in the “multiplex” that coincidentally is in the same mass “window.”

demonstrated discordant results. Discordant specimen results were not indicative of any problematic assays within the LDT panel. Rather, these represent specimens with mutations that were detected by the LDT but not the authority laboratory because these mutations were not on the authority laboratory’s panel, or vice versa. Five of the 386 specimens (1.3%) demonstrated discrepant results. In each case, the discrepant result was observed in the A455E assay portion of the LDT. Every normal and mutant mass spectrum for every assay for every specimen (386 specimens ⫻ 108 assays times two alleles per assay ⫽ 83,376) was examined by at least two medical technologists. The software provides many metrics for quantitative analysis, eg, signal to noise Table 3.

ratio, peak height, peak area, percent extension, etc, which are combined to generate an objective “peak probability.” Peak probability for recorded genotypes was highly reproducible; one example (for the ⌬F508 assay within the LDT) is shown in Table 4.

Discussion The MALDI-TOF detection system described here, using a 108-member CF carrier screening panel as a model, was shown to be reliable and accurate in the last phase of an analytical validation exercise undertaken just before offering this LDT to the ordering physician community.

Results of Validation Study by Authority Laboratory (concordance, discordance, and discrepancies)

Institution no.

Fraction of concordant specimens detected by LDT (%)

Fraction of discordant specimens uncovered during validation (%)

No. discrepant (%)

1 2 3 4 5 6 7 8 9 Totals Totals†

91/99 (92) 21/25 (84) 29/34 (85) 91/96 (95) 19/20 (95) 26/26 (100) 48/50 (96) 10/12 (83) 21/24 (88) 356/386 (92.2) 357/386 (92.5)

7 (7) 4 (16) 4† (12) 3 (3) 1 (5) 0 (0) 2 (4) 2 (17) 2 (8) 25 (6.5) 24 (6.2)

1* (1) 0 (0) 1* (3) 2‡ (2) 0 (0) 0 (0) 0 (0) 0 (0) 1* (4) 5 (1.3) 5 (1.3)

*A455E. † One of these four was a transcription error; the results were clearly in agreement before insertion of incorrect result into report sent to one of the authority laboratories. This discordance was immediately resolved and is reflected in the Totals line marked with a dagger (one additional concordant specimen and one fewer discordant specimen). ‡ Per the authority laboratory’s director, two aliquots of the same specimen were aliquoted into two tubes and given two separate identification numbers, so this discrepancy is arguably a single event that was “duplicated”; in both cases it is the failed A455E assay within the LDT, which was “repaired” shortly after “launch” of the test.


Table 4.

Peak Probability for 59 ⌬F508 Heterozygous Mutant Specimens

Sample ID

Peak 1 probability

Peak 2 probability

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

0.9961 0.9952 0.9949 0.9950 0.9955 0.9951 0.9957 0.9957 0.9957 0.9953 0.9957 0.9959 0.9957 0.9952 0.9944 0.9900 0.9903 0.9896 0.9903 0.9894 0.9887 0.9899 0.9892 0.9905 0.9900 0.9903 0.9913 0.9909 0.9886 0.9957 0.9950 0.9956

0.9938 0.9946 0.9941 0.9949 0.9953 0.9963 0.9960 0.9954 0.9957 0.9950 0.9954 0.9951 0.9956 0.9945 0.9956 0.9889 0.9895 0.9908 0.9897 0.9890 0.9913 0.9907 0.9899 0.9905 0.9890 0.9914 0.9898 0.9899 0.9880 0.9964 0.9947 0.9952

Sample ID

Peak 1 probability

Peak 2 probability

33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59

0.9944 0.9949 0.9955 0.9948 0.9951 0.9953 0.9943 0.9952 0.9948 0.9959 0.9953 0.9953 0.9950 0.9945 0.9944 0.9947 0.9952 0.9967 0.9955 0.9948 0.9950 0.9951 0.9944 0.9951 0.9949 0.9946 0.9953

0.9957 0.9945 0.9954 0.9950 0.9959 0.9955 0.9954 0.9954 0.9959 0.9957 0.9947 0.9954 0.9942 0.9934 0.9942 0.9934 0.9944 0.9942 0.9955 0.9941 0.9952 0.9948 0.9945 0.9955 0.9947 0.9952 0.9960

Average and SD for peak 1 probability ⫽ 0.9939 ⫾ 0.0023 Average and SD for peak 2 probabilityn ⫽ 0.9938 ⫾ 0.0023

To determine the genotype of a specimen using the mass spectrometry-based technique described in this article, the peaks generated by the extension products for both the normal and mutant alleles were visually examined by at least two medical technologists in each case. The software, Typer version 4.0, described in Materials and Methods uses several quantitative metrics, eg, signal to noise ratio, peak shape, peak offset, peak width, etc., to derive a peak probability score. The data in this table demonstrate excellent peak probability reproducibility within one assay (⌬F508) for a 59sample subset of the 386 specimens analyzed in this study. These data are typical of peak probability values observed for all assays for all specimens in the study.

That a complex system like formulation of a CF carrier screening panel was successfully used as a model serves to demonstrate the flexibility of the platform. By choosing biochemically amendable targets (in this case, naturally including the ACMG panel of 23 recommended CF mutations) and designing appropriate associated amplification reactions and single base extension probes for allele interrogation, we demonstrated at least a second example of a medium density mutation or polymorphism panel that may be assembled for multiplex detection.10 The process of introducing an LDT may include feasibility assessment, optimization, verification, and validation as a subset of design control15 or simply verification and validation,6,7,17 the latter of which is mandated by the CAP Laboratory Accreditation Program. We opted for a more rigorous process that more closely mimicked design control. Since CF carrier screening is an established application, a sufficient demonstration of feasibility consisted of pilot “proof of concept” experiments (data not shown) and the documentation of an appropriate literature search for both panel content and suitability of mass spectrometry as a multiplex detection platform. Significant assay design was performed during the optimization phase to perfect the test and the multiplex amplification

and SBE reactions needed to cover all 108 targets chosen. This optimized test was confirmed as a working test during the verification phase17 when, in addition to documentation of accuracy, precision, sensitivity, and specificity (data not shown), the test was used to confirm the genotypes of specimens in a “Challenge Panel” provided in a “blinded” fashion by one of the authors (W.E.H.).18 It is arguable if “blinded” specimens were necessary for verification but their inclusion added rigor to the process and confidence in our design. Finally, substantiation that the optimized and verified test performed satisfactorily with authentic clinical specimens was established in the fraction of the validation phase reported here. (We refer specifically only to the portions of the CAP principles and practices on test validation that shed light on “how many specimens [must be] run to validate a given test” and that “a new laboratory test is ready to be introduced when it is demonstrably equivalent to or better than what was already available for supporting the care of a patient.”).7 A public database was used to identify clinical laboratories performing CF screening, and these laboratories’ directors were canvassed for their willingness to provide specimens in a blinded fashion. Three hundred eighty-six


Table 5.

Residual Risk of CF after Screening Negative for the 108 Targets in the Described LDT

Racial or ethnic group Ashkenazi Jewish Non-Hispanic Caucasian Hispanic American African American Asian American

Carrier frequency (ACOG 2005)

LDT detection rate

Residual risk after ACMG 23, no family history

Residual risk, no family history (LDT)

Affected sibling (prior risk 2⁄3), residual risk (LDT)

Affected 1⁄2-sibling/ niece/nephew (prior risk 1⁄2) (LDT)

Affected aunt/uncle (prior risk1⁄3) (LDT)

Affected cousin (prior risk 1⁄4) (LDT)

1/24

99%

1/400

1/2301

1/51

1/101

1/201

1/301

1/25

92%

1/208

1/301

1/7

1/14

1/26

1/39

1/46

83%

1/164

1/266

1/4

1/7

1/13

1/19

1/65

81%

1/186

1/338

1/4

1/6

1/12

1/17

1/94

49%

1/184

1/183

1/2

1/3

1/5

1/7

specimens from nine contributing authority laboratories were used for this study. Results for all specimens were unknown to our laboratory in compliance with suggestions for test validation.7 Results generated during this study were “unblinded” by comparison with results provided by authority laboratory directors. A high level of concordance (92.5%) was observed (Table 3). Twentyfour (6.2%) of the 386 specimens demonstrated discordant results, not because of any problem with the test but rather simply because the contents of the mutation panels offered by the independent authority laboratories did not match the content of the LDT described. Thus, for example, a mutation reported by our laboratory that was marked discordant by the authority laboratory director due to lack of agreement but, as it turned out, was discordant because the authority laboratory did not include that particular mutation in its panel was immediately “resolved.” Still, since these mutations were present only in our laboratory’s panel, they were not truly “validated” by comparison with an authority laboratory result. We only felt comfortable labeling them “concordant” on resolution by sequencing. All discordant specimens were sequenced and shown to harbor the mutation detected in this study (data not shown). Thus, the resolved concordance rate was 98.7%. We detected 51 of the 108 targets in our panel during this study. To validate each assay in the panel, the 57 not observed during this study, which might take months or years to appear in submitted patient specimens, will be confirmed by sequencing before issuing a clinical laboratory test result report the first time, but not each subsequent time, they are observed. The value of this part of the process needed to launch an LDT (is “a new laboratory test . . . ready to be introduced . . . [as] demonstrably equivalent?)7 was demonstrated by the one mutation with discrepant results that was uncovered, A455E (c.1364C⬎A). Several specimens (Table 3) illuminated a failure of the A455E assay within the LDT. These discrepancies were resolved by genotyping using a Food and Drug Administration-cleared CF mutation detection test in the laboratory of one of the authors (W.E.H.); in the case of each specimen with a discrepant result, the presence of A455E (c.1364C⬎A) was confirmed. On further investigation of the assay spectra for A445E (normal and heterozygotes), we discovered that a nonspecific extension product was being produced as a result of homology of amplification primers to a pseudo-

gene on chromosome 20. This product was previously observed in spectra during test optimization, where it had been discovered that there was significant sequence homology with the A455E assay’s forward PCR primer and the pseudogene. We hypothesized that this nonspecific PCR product was preferentially being amplified over the desired A455E amplicon. Furthermore, this nonspecific product also had homology with the A455E extension primer, ultimately distorting the assay’s ability to detect the mutant peak in the case of a heterozygote. This nonspecific peak in the A455E assay spectrum did not empirically interfere with our ability to detect the normal spectral peak but it apparently deprived the reaction of reagents (primers, nucleotides) that would otherwise contribute to a more robust mutant peak. The assay’s primers, at that point in time during optimization, were redesigned and the problem was not encountered during verification. An inadvertent reagent switch between verification and validation was the root cause of the problem. Ultimately, the correct reagents were added back to the LDT, validated for successful detection of A455E (c.1364C⬎A) heterozygosity (data not shown), and these reagents are used in the mature test. Although the scope of this report is meant to emphasize the platform’s suitability in an LDT, the relatively large number of mutations and variants assembled for this CF panel indeed provide enhanced coverage for various ethnic groups when compared with the standard 23 mutation ACMG panel. Estimated residual risk calculations for individuals shown to be negative for all mutations and variants in the panel are shown in Table 5. High density arrays are appropriate for biomarker discovery. Low density platforms, eg, real-time PCR, are appropriate for interrogation of a small number of relevant targets. As we develop our knowledge of tumor biology, pharmacogenetics, biochemical pathways, and other complex processes, it seems clear we are moving toward a “middle ground” for the density of targets that must be interrogated to obtain an answer to a clinical question, ie, “clinically appropriate density.” Examples are abundant.8 –10,19 –23 This study demonstrated the suitability of mass spectrometry for use in LDTs requiring panel density of over 100 targets. Test customization occurred during design of the analytical phase of the target nucleic acids and mass spectrometry served as the tool to differentiate between resolved normal and mutant (or polymorphic) alleles.


Finally, question MOL.34229 in the CAP Laboratory Accreditation Inspection Checklist reads, “For qualitative tests, are positive . . . controls run for each assay, when available, appropriate, and practical?” A note to the question states: “[i]deally, one should use a positive control for each analyte in each run. However, in some circumstances such as in a large mutation panel for cystic fibrosis, this procedure is not practical. One way to address this situation is to rotate positive controls in a systematic fashion and at a frequency determined by the director.” We report here a test in which specific analytes (with an extended base corresponding to a normal, polymorphic, or mutant allele) are generated in a 384-well plate and then spotted onto a medium 384-density array for ionization and analysis by mass spectrometry. To interrogate all 108 targets in this LDT, positive controls are spread among 20 wells of the microtiter plate and 20 matrix pads on the array (chip), representing only 5.2% (20 of 384) of the array. This approach, therefore, provides an economical option to eliminate “rotation” of controls in CFTR (or other) genotyping tests; each assay (mutation or variant) control may be readily included with each test run.

Acknowledgments We thank Lyle Rawlings, Kyle Ingersoll, Anna Van Agtmael, Molly Dobb, Carlee Koessel, and Shirin Fitzgerald for excellent technical assistance. We thank the following clinical laboratory directors for generously providing blinded validation specimens: Drs. Laila Mnayer, Kejian Zhang, Hanna Rennert, Madhuri Hegde, Julie Jones, Julie Gastier-Foster, Carolyn Sue Richards, Marcy Hoffmann, and Maurizio Dalle Carbonare. We thank Dr. Frank Chervenak for independently correlating our LDT results with those of the independent laboratories providing samples for blinded testing. Lastly, we thank Jo Ann Spillman for excellent assistance in the preparation of this article.

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