A Droplet Digital PCR Method for Severe Combined Immunodeficiency Newborn Screening
2017; Elsevier BV; Volume: 19; Issue: 5 Linguagem: Inglês
10.1016/j.jmoldx.2017.05.011
ISSN1943-7811
AutoresNoemi Vidal‐Folch, Dragana Milosevic, Ramanath Majumdar, Dimitar Gavrilov, Dietrich Matern, Kimiyo Raymond, Piero Rinaldo, Silvia Tortorelli, Roshini S. Abraham, Devin Oglesbee,
Tópico(s)Blood disorders and treatments
ResumoSevere combined immunodeficiency (SCID) benefits from early intervention via hematopoietic cell transplantation to reverse T-cell lymphopenia (TCL). Newborn screening (NBS) programs use T-cell receptor excision circle (TREC) levels to detect SCID. Real-time quantitative PCR is often performed to quantify TRECs in dried blood spots (DBSs) for NBS. Yet, real-time quantitative PCR has inefficiencies necessitating normalization, repeat analyses, or standard curves. To address these issues, we developed a multiplex, droplet digital PCR (ddPCR) method for measuring absolute TREC amounts in one DBS punch. TREC and RPP30 levels were simultaneously measured with a Bio-Rad AutoDG and QX200 ddPCR system. DBSs from 610 presumed-normal, 29 lymphocyte-profiled, and 10 clinically diagnosed infants (1 X-linked SCID, 1 RAG1 Omenn syndrome, and other conditions) were tested. Control infants showed 14 to 474 TREC copies/μL blood. SCID infants, and other TCL conditions, had ≤15 TREC copies/μL. The ddPCR lower limit of quantitation was 14 TREC copies/μL, and the limit of detection was 4 TREC copies/μL. Intra-assay and interassay imprecision was <20% CV for DBSs at 54 to 60 TREC copies/μL. Testing 29 infants with known lymphocyte profiles resulted in a sensitivity of 88.9% and a specificity of 100% at TRECs <20 copies/μL. We developed a multiplex ddPCR method for the absolute quantitation of DBS TRECs that can detect SCID and other TCL conditions associated with absent or low TRECs and validated this method for NBS. Severe combined immunodeficiency (SCID) benefits from early intervention via hematopoietic cell transplantation to reverse T-cell lymphopenia (TCL). Newborn screening (NBS) programs use T-cell receptor excision circle (TREC) levels to detect SCID. Real-time quantitative PCR is often performed to quantify TRECs in dried blood spots (DBSs) for NBS. Yet, real-time quantitative PCR has inefficiencies necessitating normalization, repeat analyses, or standard curves. To address these issues, we developed a multiplex, droplet digital PCR (ddPCR) method for measuring absolute TREC amounts in one DBS punch. TREC and RPP30 levels were simultaneously measured with a Bio-Rad AutoDG and QX200 ddPCR system. DBSs from 610 presumed-normal, 29 lymphocyte-profiled, and 10 clinically diagnosed infants (1 X-linked SCID, 1 RAG1 Omenn syndrome, and other conditions) were tested. Control infants showed 14 to 474 TREC copies/μL blood. SCID infants, and other TCL conditions, had ≤15 TREC copies/μL. The ddPCR lower limit of quantitation was 14 TREC copies/μL, and the limit of detection was 4 TREC copies/μL. Intra-assay and interassay imprecision was <20% CV for DBSs at 54 to 60 TREC copies/μL. Testing 29 infants with known lymphocyte profiles resulted in a sensitivity of 88.9% and a specificity of 100% at TRECs 80% for SCID infants treated by hematopoietic stem cell transplantation within the first few months of life with, or without, resolved infections are in stark contrast to reported survival rates of only 50% for older infants with an active infection during transplant. Hence, early diagnosis of SCID is vital for achieving improved treatment success. Given the severity of SCID-related symptoms and improved outcomes after an early diagnosis, newborn screening (NBS) fulfills classic Wilson-Jungner screening criteria, and SCID was deemed a suitable candidate for NBS by the US Department of Health and Human Services, per a letter issued on February 25, 2010, to the Secretary of Health and Human Services. Early detection and diagnosis is currently available by newborn screening programs in most US states.1Kwan A. Abraham R.S. Currier R. Brower A. Andruszewski K. Abbott J.K. et al.Newborn screening for severe combined immunodeficiency in 11 screening programs in the United States.JAMA. 2014; 312: 729-738Crossref PubMed Scopus (468) Google Scholar Newborn screening for SCID has also been evaluated by studies at several other locations in North America, Europe, and Asia.4Jilkina O. Thompson J.R. Kwan L. Van Caeseele P. Rockman-Greenberg C. Schroeder M.L. Retrospective TREC testing of newborns with severe combined immunodeficiency and other primary immunodeficiency diseases.Mol Genet Metab Rep. 2014; 1: 324-333Crossref PubMed Scopus (18) Google Scholar, 5Audrain M. Thomas C. Mirallie S. Bourgeois N. Sebille V. Rabetrano H. Durand-Zaleski I. Boisson R. Persyn M. Pierres C. Mahlaoui N. Fischer A. 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Changes in thymic function with age and during the treatment of HIV infection.Nature. 1998; 396: 690-695Crossref PubMed Scopus (1577) Google Scholar Low TREC amounts mirror concurrent T-cell lymphopenia (TCL), and neonatal dried blood spot (DBS) TREC levels can be used to detect impaired T-cell development, and SCID by newborn screening.13Baker M.W. Grossman W.J. Laessig R.H. Hoffman G.L. Brokopp C.D. Kurtycz D.F. Cogley M.F. Litsheim T.J. Katcher M.L. Routes J.M. Development of a routine newborn screening protocol for severe combined immunodeficiency.J Allergy Clin Immunol. 2009; 124: 522-527Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 14Routes J.M. Grossman W.J. Verbsky J. Laessig R.H. Hoffman G.L. Brokopp C.D. Baker M.W. Statewide newborn screening for severe T-cell lymphopenia.JAMA. 2009; 302: 2465-2470Crossref PubMed Scopus (169) Google Scholar After a positive NBS result, diagnostic follow-up is required to determine whether the infant has typical SCID, or another form of TCL, and to establish a specific diagnosis.15Picard C. Al-Herz W. Bousfiha A. Casanova J.L. Chatila T. Conley M.E. Cunningham-Rundels C. Etzioni A. Holland A. Klein C. Nonoyama S. Ochs H.D. Oksenhendler E. Puck J.M. Sullivan K.E. Tang M.L. Franco J.L. Gaspar H.B. Primary immunodeficiency diseases: an update on the classification from the International Union of Immunological Societies Expert Committee for Primary Immunodeficiency 2015.J Clin Immunol. 2015; 35: 696-726Crossref PubMed Scopus (515) Google Scholar SCID can be detected in DBSs using real-time quantitative PCR (qPCR) for TRECs as a single-plex,16Chien Y.H. Chiang S.C. Chang K.L. Yu H.H. Lee W.I. Tsai L.P. Hsu M.H. Hwu W.L. 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Janzi M. Winiarski J. Sack U. Pan-Hammarström Q. Borte M. Hammarström L. Neonatal screening for severe primary immunodeficiency diseases using high-throughput triplex real-time PCR.Blood. 2012; 119: 2552-2555Crossref PubMed Scopus (163) Google Scholar or combined with targets for other conditions, such as an SMN1 exon 7 deletion for spinal muscular atrophy.22Taylor J.L. Lee F.K. Yazdanpanah G.K. Staropoli J.F. Liu M. Carulli J.P. Sun C. Dobrowolski S.F. Hannon W.H. Vogt R.F. Newborn blood spot screening test using multiplexed real-time PCR to simultaneously screen for spinal muscular atrophy and severe combined immunodeficiency.Clin Chem. 2015; 61: 412-419Crossref PubMed Scopus (56) Google Scholar Presently, there is no standardized SCID newborn screening method and the ideal newborn screening assay for SCID must be able to distinguish specimens with absent, or low, TREC content from those with insufficient DNA yield. The variability in newborn DBS specimen collection adds to the challenge of distinguishing samples with abnormal TREC levels. Because most SCID assays are based on PCR amplification, low results could be because of artifacts, such as an inadequate DBS sample, failure to elute a sufficient DNA amount, or the presence of a DNA polymerase inhibitor. To ensure that a low TREC result is not because of an artifact (causing a false-positive screening result), most TREC assays include simultaneous, or sequential, amplification of a conserved genomic sequence, like the ribonuclease P gene, RPP30, or the β-actin gene, ACTB.13Baker M.W. Grossman W.J. Laessig R.H. Hoffman G.L. Brokopp C.D. Kurtycz D.F. Cogley M.F. Litsheim T.J. Katcher M.L. Routes J.M. Development of a routine newborn screening protocol for severe combined immunodeficiency.J Allergy Clin Immunol. 2009; 124: 522-527Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 20Gerstel-Thompson J.L. Wilkey J.F. Baptiste J.C. Navas J.S. Pai S.Y. Pass K.A. Eaton R.B. Comeau A.M. High-throughput multiplexed T-cell-receptor excision circle quantitative PCR assay with internal controls for detection of severe combined immunodeficiency in population-based newborn screening.Clin Chem. 2010; 56: 1466-1474Crossref PubMed Scopus (75) Google Scholar Detection of a reference gene within the expected range indicates that the tested specimen is amplifiable and TREC quantification is reliable. Another sensitive method to detect low concentrations of a DNA target within a single analysis is droplet digital PCR (ddPCR).23Hudecova I. Digital PCR analysis of circulating nucleic acids.Clin Biochem. 2015; 48: 948-956Crossref PubMed Scopus (133) Google Scholar With ddPCR, a single DNA sample is partitioned into thousands of uniform-size droplets and subsequently amplified by standard PCR, and a signal from a fluorescently labeled probe within each droplet is recorded as either positive or negative, depending on the presence or absence of a target's PCR product. This results in specific and sensitive fluorescent signals derived from thousands of individual droplets containing target product. Negative droplets do not provide a fluorescent signal as they are void of target DNA amplicon. The application of a Poisson distribution calculation across all droplets permits an absolute concentration determination for a DNA target and eliminates the need for standard curves of reference standards or endogenous controls. Thousands of replicate measurements of each sample occur during each run. Last, the application of differentiated labeled fluorescent probes allows for multiplexing PCR targets, such as TRECs and RPP30, within a single DNA tube and from the same DBS punch. Given the efficiencies promised by ddPCR, we designed a ddPCR assay to simultaneously measure the absolute level of TRECs and RPP30. Validation of this method is described herein. Infant and cord blood specimens were collected with Mayo Clinic Institutional Review Board approval. A population-based TREC reference range was determined from 610 (541 full-term and 69 preterm) residual, anonymized DBSs collected onto Whatman 903 Protein Saver Cards (GE Health Care, Pittsburgh, PA) whose demographics are shown in Table 1. Infants with a gestational age <37 weeks were considered preterm. DBSs were also prepared from 10 infants diagnosed with X-linked SCID, Omenn syndrome, idiopathic TCL, chromosome 22q11.2 deletion-negative TCL, DiGeorge syndrome, CHARGE (coloboma of the eye, heart abnormalities, coanal atresia, retardation of growth and development, genitourinary anomalies, ear anomalies and deafness) syndrome, ataxia telangiectasia, and cartilage hair hypoplasia, whose demographic data are listed in Table 1 and TREC results in Table 2. Moreover, 29 blood samples submitted to the Mayo Clinic for follow-up testing, after NBS results, were anonymized and analyzed with data shown in Table 3.Table 1Demographics of Presumed Normals and Low TREC SubjectsCohort/specimenSexAge at collection, daysGestational age, weeksBirth weight, gFull-term infants (n = 541)272 females, 264 males, 5 not provided1–133 (3)37–41 (38)1660–4680 (3020)Preterm infants (n = 69)30 Females, 39 males1–74 (7)27–36 (36)1350–3885 (2290)DBS001Male15NA4069DBS002Female41NANADBS003Male8404454DBS004Male8NA3852DBS005Male6373300DBS006Female8NANADBS007Female8NANADBS008Male6NANADBS009Male22NANADBS010Male12NANAMedian values are shown in parenthesis.NA, not available; TREC, T-cell receptor excision circle. Open table in a new tab Table 2SCID Screen-Positive CohortSpecimenWhole blood results (reference range)∗Reference ranges listed are age specific.Clinical diagnosisDBS results (reference range)∗Reference ranges listed are age specific.TREC copies/106 CD3 T cells (≥6794)CD3+ T cells, cells/μL (1484–5327)CD4+ T cells, cells/μL (733–3181)CD8+ T cells, cells/μL (370–2555)CD19+ B cells, cells/μL (370–2306)CD16+56+ NK cells, cells/μL (43–256)CD45RA % of CD4 T cellsCD45RO % of CD4 T cellsCD4+31+45RA+ % of CD4+45RA+ T cellsTREC copies/μL blood (<20)RPP30 copies/μL blood (≥1500)DBS001122010447293198421480801552Idiopathic T-cell lymphopenia1014,550DBS00299891268622920215884115722q11Deletion-negative TCL523,550DBS0032201814429379417549732046Idiopathic T-cell lymphopenia1517,775DBS004219212939863141226446543082DiGeorge syndrome613,413DBS005124134424894252159681257DiGeorge syndrome410,500DBS0061090174116427951681504176Cartilage hair hypoplasia<411,925DBS007569814369144951131418593963Ataxia telangiectasia117513DBS008<524318325420940X-linked SCID<43013DBS0091836382307691006317742647CHARGE syndrome<45438DBS010<54244081521420<1950RAG1 SCID/Omenn syndrome<48150CHARGE, coloboma of the eye, heart abnormalities, coanal atresia, retardation of growth and development, genitourinary anomalies, ear anomalies and deafness; DBS, dried blood spot; NK, natural killer; SCID, severe combined immunodeficiency; TCL, T-cell lymphopenia; TREC, T-cell receptor excision circle.∗ Reference ranges listed are age specific. Open table in a new tab Table 3SCID Newborn Screen Follow-Up CohortSampleSexAge, daysOriginal NBS resultWhole blood results (reference range)DBS results (reference range)TREC copies/106 cells (≥6794)CD3+ T cells (1484–5327)CD4+ T cells (733–3181)CD8+ T cells (370–2555)Lymphocyte-based interpretationTREC copies/μL blood (≥20)RPP30 copies/μL blood (≥1500)DBS017Male5Abnormal4560409624931444Normal19022,188DBS019Male10Abnormal562834632718778Normal9819,863DBS020Female18Abnormal7037407429601124Normal20121,700DBS021Male18Abnormal322731892646518Normal10617,988DBS022Male9Abnormal5233309720481036Normal11614,763DBS023Male9Abnormal580934182649760Normal14819,350DBS024Male17Abnormal5900366922281337Normal439000DBS025Female64Abnormal98301091785291Normal3424,450DBS026Female48Abnormal736917941534238Normal697813DBS027Male33Abnormal315532302396795Normal8517,463DBS029Female7Abnormal51441209898270Normal2911,150DBS030Male9Abnormal49111090900199Normal5035,375DBS031Male115Abnormal467025111775595Normal10117,175DBS032Female9Abnormal795720391444583Normal9425,488DBS037Female6Abnormal788818841658251Normal7949,500DBS054Female21Abnormal435918311297516Normal2411,375DBS055Male62Abnormal8391508143TCL<46238DBS058Male15NA22,188461234241115Normal14517,050DBS059Female17NANA20221611412Normal8014,625DBS060Male32NANA717567152TCL∗Final TCL diagnosis was unestablished because of absent TREC copies/106 cell levels.2519,850DBS061Male65NA299821561108926TCL1412,300DBS062Female11NA16,4061266843418Normal3313,150DBS063Female27Abnormal<596389824SCID<45025DBS064Male53NA74871135687362iTCL1154,875DBS065Male5Abnormal17,552513912Secondary TCL†Final secondary TCL diagnosis was unconfirmed as CD4 recent thymic emigrant % and clinical history were unavailable.47388DBS066Male14Abnormal7469956672286TCL1510,488DBS067Female37NA62471167037TCL419,275DBS068Female46Abnormal294832127742Leaky SCID<47438DBS069Female10Abnormal22,56627512288451Normal15519,538iTCL, idiopathic TCL; NA, not available; NBS, newborn screening; SCID, severe combined immunodeficiency; TCL, T-cell lymphopenia; TREC, T-cell receptor excision circle.∗ Final TCL diagnosis was unestablished because of absent TREC copies/106 cell levels.† Final secondary TCL diagnosis was unconfirmed as CD4 recent thymic emigrant % and clinical history were unavailable. Open table in a new tab Median values are shown in parenthesis. NA, not available; TREC, T-cell receptor excision circle. CHARGE, coloboma of the eye, heart abnormalities, coanal atresia, retardation of growth and development, genitourinary anomalies, ear anomalies and deafness; DBS, dried blood spot; NK, natural killer; SCID, severe combined immunodeficiency; TCL, T-cell lymphopenia; TREC, T-cell receptor excision circle. iTCL, idiopathic TCL; NA, not available; NBS, newborn screening; SCID, severe combined immunodeficiency; TCL, T-cell lymphopenia; TREC, T-cell receptor excision circle. Three levels of controls were prepared as DBSs to evaluate the performance characteristics of the TREC assay: i) an SCID-like sample, with TREC content lower than the expected range for newborns, but normal RPP30 levels; ii) a borderline negative control with TREC content adjacent to the assay's threshold between normal and abnormal (decreased but not absent TREC), and normal RPP30 levels; iii) a negative control with TREC content within the expected newborn range, and normal RPP30 levels. In addition, DBSs were prepared for stability, accuracy, linearity, imprecision, clinical sensitivity, and specificity studies by spotting 75 μL of EDTA whole blood onto Whatman 903 cards and drying overnight at room temperature. DBSs were stored at −20°C with desiccant in sealed plastic bags. Positive-control DBS cards were made from anonymized adult EDTA whole blood, and negative-control DBS cards were made from donor umbilical cord blood, both collected with institutional review board approval. Last, 30 DBSs were obtained from the CDC's Newborn Screening Quality Assurance Program for TREC Analysis in DBS to assess performance against other laboratories using qPCR or an EnLite Neonatal TREC kit (Perkin Elmer, Waltham, MA). Every quarter, the CDC distributes a panel of five unknown DBS specimens to domestic, international, and manufacturer laboratories to analyze TREC content in peripheral blood. An overview of the ddPCR assay workflow is shown in Figure 1. A DNA extraction protocol was established for this study. Briefly, a single 3.2-mm disk was punched from DBSs into a semiskirted PCR 96-well plate (Eppendorf, Hamburg, Germany). DBS disks were washed twice by adding 120 μL Generation DNA Purification solution (Qiagen, Valencia, CA) and heating to 50°C for 30 minutes. After washing, 120 μL Generation DNA Elution buffer (Qiagen) was added, and the plate was incubated at room temperature for 15 minutes. After removing the Generation DNA solution, 15 μL of water was distributed per well, and the plate was heated to 99°C for 25 minutes to release DNA. Last, the plate was stored overnight at 4°C, followed by a high-speed centrifugation at 3500 × g for 10 minutes to remove debris. The ddPCR TREC primers and probe sequences were the same as those described by Douek et al.11Douek D.C. Vescio R.A. Betts M.R. Brenchley J.M. Hill B.J. Zhang L. Berenson J.R. Collins R.H. Koup R.A. Assessment of thymic output in adults after haematopoietic stem-cell transplantation and prediction of T-cell reconstitution.Lancet. 2000; 355: 1875-1881Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar RPP30 primers and probes were newly designed for this assay (Table 4). Both TREC and RPP30 ddPCR assay mixes were custom synthesized by IDT (Coralville, IA). Amplicon sizes for the expected PCR products for TREC and RPP30 were 109 and 65 bp, respectively. The ddPCR mixture was assembled by an automated liquid handler system. Reaction mixtures of 22 μL were prepared in semiskirted PCR 96-well plates with 11 μL of 2× ddPCR Supermix for probes (no dUTP) (Bio-Rad, Pleasanton, CA), 1.1 μL of TREC assay mix, 1.1 μL of RPP30 assay mix, and 8.8 μL of DBS DNA. The final concentrations of primers and probes in the reaction were 900 and 250 nmol/L, respectively. Multiwell plates were sealed with foil at 180°C for 4 seconds using a Plate Sealer (Bio-Rad), vortexed briefly, centrifuged, and placed on the automated droplet generator, AutoDG (Bio-Rad), where the generation of droplets occurred by loading 20 μL of Reaction Mix and 70 μL of droplet oil into a DG-32 cartridge (Bio-Rad). After droplet generation, plates were resealed with the Plate Sealer. PCR amplification was performed on a Veriti Thermal Cycler (Applied Biosystems, Foster City, CA) using end point PCR conditions: 95°C for 10 minutes, followed by 40 cycles of denaturation at 94°C for 30 seconds, annealing and extension at 58°C for 1 minute, and a final extension step at 98°C for 10 minutes. Plates were stored at 4°C until read on a QX200 Droplet Reader (Bio-Rad).Table 4Primers and Probes for ddPCRReagentSequenceTREC Forward primer5′-CACATCCCTTTCAACCATGCT-3′ Reverse primer5′-GCCAGCTGCAGGGTTTAGG-3′ Probe5′-FAM-ACACCTCTGGTTTTTGTAAAGGTGCCCACT-ZEN/IBFQ-3′RPP30 Forward primer5′-AGATTTGGACCTGCGAGCG-3′ Reverse primer5′-GAGCGGCTGTCTCCACAAGT-3′ Probe5′-HEX-TTCTGACCTGAAGGCTCTGCGCG-ZEN/IBFQ-3′TREC, T-cell receptor excision circle. Open table in a new tab TREC, T-cell receptor excision circle. QuantaSoft software version 1.7.4 (Bio-Rad) was used for droplet cluster classification, and multiwell threshold was applied on a plate-by-plate basis. For each fluorophore, the fraction of positive droplets was fitted into a Poisson distribution equation, thereby providing absolute quantification of TREC and RPP30 PCR products per well without a standard curve. Rejection criteria for excluding a result from the data summary included a clog detected by the QX200 reader or the assay threshold) or SCID-screen positive (TREC content < the threshold). The detection capability for this assay was established following guidelines by the Clinical and Laboratory Standards Institute.24Clinical and Laboratory Standards Institu
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