BACKGROUND: Real-time quantitative PCR (qPCR) targeting a specific marker of functional T cells, the T-cell–receptor excision circle (TREC), detects the absence of functional T cells and has a demonstrated clinical validity for detecting severe combined immunodeficiency (SCID) in infants. There is need for a qPCR TREC assay with an internal control to monitor DNA quality and the relative cellular content of the particular dried blood spot punch sampled in each reaction. The utility of the qPCR TREC assay would also be far improved if more tests could be performed on the same newborn screening sample.
METHODS: We approached the multiplexing of qPCR for TREC by attenuating the reaction for the reference gene, with focus on maintaining tight quality assurance for reproducible slopes and for prevention of sample-to-sample cross contamination. Statewide newborn screening for SCID using the multiplexed assay was implemented, and quality-assurance data were recorded.
RESULTS: The multiplex qPCR TREC assay showed nearly 100% amplification efficiency for each of the TREC and reference sequences, clinical validity for multiple forms of SCID, and an analytic limit of detection consistent with prevention of contamination. The eluate and residual ghost from a 3.2-mm dried blood spot could be used as source material for multiplexed immunoassays and multiplexed DNA tests (Multiplex Plus), with no disruption to the multiplex TREC qPCR.
CONCLUSIONS: Population-based SCID newborn screening programs should consider multiplexing for quality assurance purposes. Potential benefits of using Multiplex Plus include the ability to perform multianalyte profiling.
Severe combined immunodeficiency (SCID)5 is a term that describes a spectrum of primary immunodeficiencies that comprise about 14 independent genetic conditions (1, 2), all of which yield severe defects in cellular and humoral immunity. The set of conditions associated with SCID holds promise to be ideal for identification by population-based newborn screening (NBS): SCID has a clear case definition, near uniform mortality by age 1 if untreated, and the potential of curative treatment by immune reconstitution if intervention is early (3). NBS for SCID has been the subject of national workshops convened since 2001 (4, 5), and although SCID was not on the list of 29 conditions recommended by the American College of Medical Genetics for a uniform national screening panel (6), it is generally believed that only the lack of a proven screening assay prevented SCID from being included in the initial recommendations. SCID was 1 of the first 2 conditions to be nominated by the Federal Secretary's Advisory Committee on Heritable Disorders and Genetic Diseases in Newborns and Children for addition to a uniform national NBS panel and evaluated by the recently established advisory committee's external evidence review workgroup. The committee recommended additional data collection on SCID after discussion relative to issues of quality assurance parameters for expansion beyond research protocols (7). SCID has now been recommended by the committee for inclusion on the uniform national screening panel (8).
The feasibility of a screening assay for detection of SCID that is applicable to the newborn sample, the NBS dried blood spot (DBS), has been investigated in a research setting by Chan and Puck (9), who recognized that the Douek assay (10) for quantifying thymic production of T cells [which measures excisional DNA products of T-cell–receptor gene rearrangements (TRECs)] could be used on the NBS DBS to measure the 1 common underlying characteristic of all SCID conditions, the complete absence or extremely low concentration of autologous or functional T cells (1). TRECs, which are numerous in newly generated infant T cells, were the analyte of choice to ensure that the SCID screening assay would not be confounded by adult T cells that are transplacentally acquired from the mother in utero through maternal engraftment, which occurs commonly in SCID patients (11). Maternally derived T cells are not newly generated and would have low TREC. The original TREC assay set the standard to be met because it showed good analytic and clinical validity, but it was lacking in some practical and quality assurance measures. The singleplex TREC assay described by Chan and Puck called for 2 aliquots or 2 “paper-punches” from each DBS, which is highly unusual for a screening test that would be applied to every sample. The original singleplex TREC assay also had an unacceptably high rate of positive screening results (1.4%) that seemed to be inconsistent with expected SCID incidence and was likely due to samples with nonamplifiable DNA. Researchers in Wisconsin used the singleplex TREC assay in the first population-based application of SCID NBS and demonstrated that the previously observed high false-positive rate could be attributed to unsatisfactory samples (12). The Wisconsin group streamlined the singleplex protocol, measuring from two 3.2-mm DBS only when the TREC assay showed numbers of TRECs below the cutoff set by the Wisconsin laboratory. According to the Wisconsin protocol, identification of samples with low or absent TRECs relies on evaluation of sample quality by demonstration of the amplifiability of a reference gene with an assay that is performed in a separate reaction from the TREC assay.
In Massachusetts, we looked at the implementation of SCID NBS (the first disorder for which NBS would require a DNA-based assay as the first tier of a screening algorithm) as an opportunity to develop a high-quality DNA platform for population-based screening. Although such a DNA platform might be used for basic genetic assays in which alleles of single-copy genes are reported, it would have to be useful for detection and quantification of extrachromosomal or low–copy-number DNA sequences. Ideally, such a platform would facilitate generation of internally controlled quantitative results for potentially disparate quantities and types of DNA targets. Such characteristics would be demanded for a TREC quantitative PCR (qPCR) that would be multiplexed with an internal reference gene, and the validation of this assay would provide a demonstration of success with the platform. The standard curve for such a quantitative assay as the TREC multiplex qPCR would have to reliably report absence and very low copy numbers of TREC to avoid false-negative results. The assay and algorithm would have to address some of the more challenging aspects of working with the newborn DBS: limited available quantity of whole blood and possible variation in cellular distribution across the filter-paper matrix. Because of the nature of the SCID screening algorithm (seeking the absence of the TREC as an indicator of risk for SCID), the assay would have to be integrated within laboratory processes that approach 100% prevention of sample-to-sample contamination. In addition, we recognized that if the DNA component of the assay could be multiplexed to markers used in, e.g., immunoassays, then multianalyte profiling would be possible and might be integrated into interpretation of the NBS result.
We report development and implementation of a multiplexed TREC qPCR assay in which both the TREC analyte and its internal control are acquired from a single sample (punch), and run in exactly the same reaction. We provide quality assurance data for population-based screening such as NBS for SCID. We also report preliminary data that demonstrate the technical capacity for incorporation and analysis of data from non-DNA analytes as well as DNA markers.
Materials and Methods
SOURCES OF INFANT AND ADULT SAMPLES
Data for this study were obtained from the following sources: (a) the residual NBS DBS from a total of 8 neonates born before implementation (2 of whom were demonstrated to have engraftment of maternal T cells), and in whom 5 different SCID conditions were previously diagnosed; (b) a DBS series collected before transplantation from 1 of these infants at an older age in whom SCID had been diagnosed and whose sample was coded for use in the New England Newborn Screening Program; (c) coded adult samples; (d) sets of deidentified residual DBS from the Massachusetts NBS program used as part of assay validation before implementation of the pilot screening; and (e) identified infants who were included after implementation of the statewide SCID NBS pilot program. All of the deidentified and coded uses of samples for validation of the assay, as well as the identified uses of samples for implementation of the SCID NBS pilot program, were approved by an institutional review board (13).
PREPARATION OF CALIBRATION CURVES
After we confirmed the concentration (using a Nanodrop spectrophotometer) of the 3891-bp plasmid containing the δRec-ΨJα TREC-specific sequences (10), we prepared 2-fold serial dilutions at 2× concentrations in diluent [Qiagen Generation Solution 2 (S2), containing tRNA at 50 mg/L (50 ng/μL)] for the 8-point calibration curve.
Likewise, human genomic DNA (Zyagen) was prepared in 2-fold serial dilutions at 2× concentrations. Each point of the 2× TREC dilution series was then combined with an equal volume of the respective point of the 2× human DNA dilution series to make a 1× calibration-curve working stock. The multiplexed calibrators from the working stock were run in quadruplicate at dilutions that bracketed empirically determined values within reference intervals from validation testing of deidentified samples. Each 8-point set of calibrators included concentrations of TREC plasmid and the ribonuclease P RNA component H1(RPPH1)6 gene, respectively, at: 39 and 625, 78 and 1250, 156 and 2500, 312 and 5000, 625 and 10 000, 1250 and 20 000, 5000 and 30 000, and 10 000 and 40 000 copies per 5 μL. The 1× working stock was divided into 24-μL single-use aliquots that were frozen at −80 °C for long-term storage or −20 °C for daily use.
DNA PREPARATION FOR TREC MULTIPLEX qPCR
Sample DNA from each sample was extracted from a single 3.2-mm DBS in a 96-well round-bottom plate (VWR). Each DBS was washed twice with 150 μL 0.5% Triton X-100 in 1× PBS buffer and once with 150 μL S2 for 10 min each at 500 rpm on a microplate shaker (VWR) at ambient temperature with aspiration of the wash in between. The washes were followed by DNA elution in 100 μL S2 at 99.5 °C while being shaken in a VorTemp 56 (Labnet) for 30 min at 500 rpm, with the use of a hermetically sealed, pierceable aluminum cover (Eppendorf) to prevent evaporation and cross-contamination. During incubation, pressure was applied to the plate seal to prevent its compromise. At no time were the plate seals peeled off.
ALTERNATIVE SAMPLE PREPARATION FOR MULTIPLEX PLUS
Before the DNA preparation described above, samples were first eluted for immunoassay by Luminex as described by Janik et al. (14). The residual ghost (the filter paper matrix and any sample left within after elution for immunoassay) from that elution was then processed as described above. (Multiplex Plus is a term from A. Comeau for the algorithm that allows both immunoassays and DNA assays to be performed on a sample acquired from the exact same sample.)
REAGENT AND SAMPLE DISTRIBUTION
All reagents were prepared in a clean room in an Airclean PCR workstation (hood). The Multidrop® Combi nL (Thermo Scientific) dispenser was used to distribute master mix reagent into 384-well plates (Applied Biosystems), which were then placed onto a Biomek 3000 (Beckman Coulter) recipient deck located in a different hood in the preamp room. The 96-well sealed plates that contained DNA eluates and preparation blanks were placed on the donor deck for distribution. The Biomek was programmed to pierce the seals of a strip of 8 wells and retrieve 5 μL DNA for delivery to the preprogrammed location in the 384-well plate. Preparation blanks were then added to preprogrammed wells on the 384-well plate. When delivery was complete, the DNA plate containing approximately 95 μL DNA that would be available for other assays was resealed. Strips containing reference DNA samples (1 sample from an adult and 2 samples from a known SCID infant) and a no-template control, and strips containing calibrators were then placed on the donor deck for distribution to preprogrammed locations across the 384-well plate.
REAL-TIME QUANTITATIVE MULTIPLEX PCR
Forward and reverse primers targeting δRec-ΨJα TREC-specific sequences were used to generate a 93-bp amplicon spanning the splice junction, with the TREC probe located just downstream from the junction. The qPCR included primers and probe for an attenuated amplification of the RNase P gene RPPH1. By limiting the amount of RNase P primer/probe reagent (TaqMan RNase P Vic Control Reagent; Applied Biosystems) that was added to the reaction mix, we prevented domination of the multiplex reaction by amplification of the more abundant RPPH1 gene. We determined the optimal volume of RNase P primer/probe reagent to be added to the multiplex reaction by adding the TREC primers and RNase P mix in a concentration matrix and selecting the optimal combination such that the Δ normalized reporter value (ΔRn) for RNase P was reduced while still attaining accurate quantification cycle (Cq) values for both targets in the TREC/attenuated RNase P reaction. The 20-μL reaction consisted of 10 μL TaqMan® Fast Universal PCR Master Mix (4367846; Applied Biosystems), 0.4 μL TaqMan RNase P Vic Control Reagent (4316844; Applied Biosystems; accession number NR_002312.1), and TREC primers and probe sequences (Applied Biosystems) located within the gene identified by accession number [NT_026437, nucleotides (forward primer) 3944229 through 3944289, and (reverse primer) 3855229 through 3855280] in the following concentrations: 8 pmol each of forward TREC primer (TGCTGACACCTCTGGTTTTTGTAA) and reverse TREC primer (GTGCCAGCTGCAGGGTTTAG), 3 pmol TREC-specific hydrolysis probe (6FAM-ATGCATAGGCACCTGC-MGB), and 5 μL of DNA eluate. Absolute qPCR was performed in an Applied Biosystems 7900 HT Real-Time PCR System in a 384-well plate (4343814; Applied Biosystems) by using a thermocycling profile as follows: denaturation of DNA for 1 cycle at 95 °C (10 min) followed by 40 cycles of 95 °C (15 s) and 60 °C (30 s). The amplified TREC product generated from several independent newborn samples was run on a 4% agarose gel and showed a single band of the appropriate size.
REAL-TIME DATA COLLECTION, DETERMINATION OF SLOPE FOR REGRESSION, AND PLATE-ACCEPTANCE CRITERIA
We collected real-time data using the Applied Biosystems Sequence Detection System (SDS) version 2.3 software. Analysis was done by manually setting the threshold at 0.08 and baseline at 3–15 for both analytes. For any single 384-well plate, we allowed elimination of up to 6 of the 32 calibration points, provided that at least 1 calibration point representing 39 TREC copies was included in the set. The acceptable ranges set for the slope were −3.10 to −3.60 with R2 ≥ 0.970 and y-intercept Cq values ≥36 and ≤41.
CONVERSION OF REAL-TIME SAMPLE DATA TO A STANDARDIZED UNIT
For each sample assayed, real-time data reported by the SDS Version 2.3 Software were converted to a universal common unit (copies per μL of whole blood) to facilitate interlaboratory standardization by dividing the SDS-reported value by 0.155: [(SDS copies/reaction) (reaction/5 μL DNA eluate) (100 μL DNA eluate/DBS) (DBS/3.1 μL whole blood)].
Data were from samples processed in the course of routine NBS for SCID between August 24, 2009 and January 1, 2010.
CALIBRATION DATA FOR TREC AND RNASE P
Regression curves for each of the TREC and RNase P calibrators (Fig. 1) showed good linearity, and the calibrators appropriately bracketed the observed values in the population (320 samples shown). Summary calibration data from 129 runs showed that the slopes and R2 were consistent with an efficiency of amplification approaching 100%; specifically, the median efficiencies for TREC and RNase P were 99% and 94%, respectively (Table 1). Data from these runs yielded a limit of detection (mean value of 129 tests of zero-TREC control + 3 SD) of 3 copies TREC/reaction or 19 copies TREC/μL whole blood. Summary Cq values for the 4 copy number values common to both of the TREC and RNase P curves (Table 2) showed that the Cq for each copy number value was consistent between each reaction in each run (note median comparison) and appropriately changed by 1 Cq for each 2-fold dilution.
Amplification curves (Fig. 2) demonstrated separation between each of the TREC calibrators when viewed alone and appropriate attenuation of the RNase P when viewed together. The distribution of Cq was not compromised, and the ΔRn for RNase P was decreased. The view of the results acquired from clinical samples in the same run showed that the reactions in clinical samples performed similarly to calibrators and that all background was well below the threshold.
The distribution of copy numbers among infants in neonatal intensive care units (NICU) and non-NICU infants showed that a higher proportion of NICU infants had low numbers of TRECs than non-NICU infants (Fig. 3). In addition, the overall number of nucleated cells appeared to be slightly lower in the NICU population than in the non-NICU population.
CALCULATION AND DETERMINATION OF RELATIVE COPY NUMBERS FOR TRECs AND REFERENCE GENE
We reasoned that a 3.2-mm DBS obtained from a neonate who was 24 to 72 hours old should have sufficient T cells [3100 T cells/μL whole cord blood (15) (3.1 μL whole newborn blood/3.2 mm DBS) = 9300 T cells/3.2 mm DBS] relative to a reference gene in the minimum subset of nucleated cells present, the leukocytes [(15 000 leukocytes/μL whole blood) (3.1 μL whole blood/3.2 mm DBS) = 45 000 leukocytes/3.2 mm DBS], to yield DNA with reproducible demonstration of TRECs, which should be present in approximately 70% to 90% of T cells from a healthy newborn (16, 17). Tests of DBS calibrators made from dilutions of known leukocyte concentrations into leukocyte-depleted blood (14) showed RNase P copy numbers consistent with expected concentrations of cells per milliliter (e.g., 2.6 × 105 ± 1.9 × 104 copies of RNase P for samples known to have 1.25 × 105 cells/mL or 2.5 × 105 copies RNase P; 6.3 × 105 ± 7.1 × 104 for samples known to have 2.5 × 105 cells/mL; and 1.7 × 106 ± 9.7 × 104 for samples known to have 5.0 × 105 cells/mL). The multiplex assay yields results that are concordant with predictions (Table 3); the mean TREC value of 2.0 × 103/μL whole blood that was observed in the non-NICU population was evidence of TREC in 50%–60% of the expected number of autologous (infant) T cells (16, 17), and the mean RNase P value of 7.2 × 104 copies/μL was within the expected range for the number of RNase P copies in the nucleated cells in a neonate. Most importantly, the results from the TREC Multiplex qPCR enabled us to distinguished known SCID infants from the general population (P < 0.0001), despite similar counts of markers for nucleated cells and despite the demonstrated maternal engraftment of T cells in 2 of the SCID infants. To our knowledge, none of the 25 609 NBS samples were obtained from SCID infants (13). The SCID infants uniformly had TREC values well below the cutoff value of 252/μL whole blood, and the majority of SCID infants in fact did not have detectable TREC (Fig. 2, Table 3). The 3 SCID infants with detectable TREC included a premature infant with high-level maternal engraftment (27 TREC), a child with purine nucleoside phosphorylase deficiency (PNP, 1.4 × 102 TREC), a genotype of SCID typically manifested by delayed loss of T-cell production (also shown in Fig. 2), and a child with SCID of unknown genotype (58 TREC).
FEASIBILITY OF MULTIPLEX PLUS
We evaluated multiple anonymous DBS samples with our Multiplex TREC using the alternative specimen preparation for Multiplex Plus. The TREC and RNase P values were similar to those observed with the routine DNA preparation. In addition, after new 3.2-mm samples from samples that had been previously evaluated by Multiplex TREC qPCR were tested by the Luminex immunoassay for CD345, the residual ghost was retested by Multiplex TREC qPCR, and the data matched the original TREC values.
The results of this investigation demonstrate the use of a multiplex qPCR assay, the Multiplex TREC qPCR, with an internal QC, the RNase P gene RPPH1, for use in population-based NBS for detection of SCID. Our data demonstrate a DNA platform (preparation, processing, and testing) that is applicable to a 3.2-mm DBS and that can be extended to include other DNA markers for NBS. In addition, we present preliminary data showing that the DNA platform can be multiplexed further to include non-DNA analytes such as CD345 as described by Janik et al. (14) for multianalyte profiling from a single 3.2 mm DBS punch.
The Multiplex TREC qPCR makes use of a DNA preparation process that yields DNA that is sufficiently clean of inhibitors to be used to quantify low copy number sequences and sufficient in DNA quantity for other DNA applications. Our sample for detection of low copy TREC used only 5 μL of the 100-μL DNA eluate, leaving a residual 95 μL for similar assays or for end-point qualitative assays of single-copy genes that by their nature require much smaller sample amounts. Although SCID is currently the only NBS condition that requires a first-tier DNA assay in our program, the availability of liquid sample for other targets under consideration for NBS permits the possibility of including other targets without the need to acquire another DBS punch sample. In addition, the use of liquid sample enhances the feasibility of using sophisticated and electronically controlled liquid-handling systems such as digital microfluidics. The low limit of detection demonstrates that our DNA platform yielded no sample-to-sample contamination, despite the high throughput of the laboratory (more than 77 000 independent samples since screening was implemented in February 2009). In addition, data from the 3 SCID infants whose samples showed low concentrations of TREC emphasizes that any assay used in population-based screening must be able to differentiate the low concentrations of naïve T cells observed in some SCID infants from concentrations within reference intervals in the general population.
A particular aspect of the multiplex TREC qPCR that might be applicable to multiplex assays for disparate targets is the attenuation of the amplification of the single-copy reference gene sequence to maximize quantification of the low copy number TREC while maintaining high precision. The assay uses a calibration curve incorporating likely relative concentrations of the TREC and reference sequence found in actual clinical samples. Although the most obvious value to the internal control is real-time validation of the quality of the sample (i.e., the DNA is amplifiable), we are investigating the analytic feasibility of using each individual sample's TREC and RNase P ratio to control for sampling error within the filter-paper matrix.
The successful implementation of the multiplex TREC qPCR has implications that go beyond a high quality SCID NBS program. This method offers the potential for streamlining some of the current second-tier DNA-based assays used in NBS and provides a strong base for the likely addition of more DNA-based assays for relevant genetic or infectious conditions. Thus, the efforts and costs required for preparing DNA from all samples and for developing algorithms for test protocols and multianalyte profiling are likely to benefit to other facets of the NBS program.
We thank G. Mushinsky, F. Bonilla, and D. Douek, J. Hale, and the Massachusetts SCID NBS Working Group.
↵5 Nonstandard abbreviations:
- severe combined immunodeficiency;
- newborn screening;
- dried blood spot;
- T-cell–receptor gene rearrangement;
- quantitative PCR;
- Qiagen Generation Solution 2;
- Δ normalized reporter value;
- quantification cycle;
- Sequence Detection System;
- neonatal intensive care unit;
- purine-nucleoside phosphorylase deficiency.
↵6 Human genes:
- ribonuclease P RNA component H1;
- interleukin 7 receptor alpha.
Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.
Authors' Disclosures of Potential Conflicts of Interest: Upon manuscript submission, all authors completed the Disclosures of Potential Conflict of Interest form. Potential conflicts of interest:
Employment or Leadership: None declared.
Consultant or Advisory Role: None declared.
Stock Ownership: None declared.
Honoraria: None declared.
Research Funding: CDC grant 1U01EH000362.
Expert Testimony: None declared.
Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.
- Received for publication February 5, 2010.
- Accepted for publication June 15, 2010.
- © 2010 The American Association for Clinical Chemistry