Improved Detection of the KIT D816V Mutation in Patients with Systemic Mastocytosis Using a Quantitative and Highly Sensitive Real-Time qPCR Assay
2011; Elsevier BV; Volume: 13; Issue: 2 Linguagem: Inglês
10.1016/j.jmoldx.2010.10.004
ISSN1943-7811
AutoresThomas Kristensen, Hanne Vestergaard, Michael Møller,
Tópico(s)Coagulation, Bradykinin, Polyphosphates, and Angioedema
ResumoThe vast majority of patients with systemic mastocytosis (SM) carry the somatic D816V mutation in the KIT gene. The KIT D816V mutation is one of the minor criteria for a diagnosis of SM according to the 2008 World Health Organization classification of myeloproliferative neoplasms. In the present study, we present a real-time qPCR assay that allows quantification of as little as 0.003% KIT D816V mutation-positive cells. A total of 61 samples from 31 cases of SM were included in the study. We detected the mutation in skin or bone marrow in 95% of the cases of SM. We demonstrate the clinical relevance of the assay by identifying as little as 0.03% mutation-positive cells in bone marrow aspirates from SM patients and calculate the analytical sensitivity of negative samples to determine the reliability of the result. We further demonstrate that this method also detects the KIT D816V mutation in peripheral blood in 81% of the mutation-positive cases with SM. The method also allows comparison of mutation-positive and mast cell fractions to determine whether the mutation is present in non-mast cells, a parameter that has recently been reported to be of prognostic importance in patients with indolent SM. Finally, the assay is suitable for use in prospective studies of the KIT D816V allele burden as a treatment endpoint in SM. The vast majority of patients with systemic mastocytosis (SM) carry the somatic D816V mutation in the KIT gene. The KIT D816V mutation is one of the minor criteria for a diagnosis of SM according to the 2008 World Health Organization classification of myeloproliferative neoplasms. In the present study, we present a real-time qPCR assay that allows quantification of as little as 0.003% KIT D816V mutation-positive cells. A total of 61 samples from 31 cases of SM were included in the study. We detected the mutation in skin or bone marrow in 95% of the cases of SM. We demonstrate the clinical relevance of the assay by identifying as little as 0.03% mutation-positive cells in bone marrow aspirates from SM patients and calculate the analytical sensitivity of negative samples to determine the reliability of the result. We further demonstrate that this method also detects the KIT D816V mutation in peripheral blood in 81% of the mutation-positive cases with SM. The method also allows comparison of mutation-positive and mast cell fractions to determine whether the mutation is present in non-mast cells, a parameter that has recently been reported to be of prognostic importance in patients with indolent SM. Finally, the assay is suitable for use in prospective studies of the KIT D816V allele burden as a treatment endpoint in SM. Mastocytosis is a heterogenous group of diseases characterized by the growth and accumulation of neoplastic mast cells.1Swerdlow S.H. Campo E. Harris N.L. Jaffe E.S. Pileri S.A. Stein H. Thiele J. Vardiman J.W. WHO classification of tumours of haematopoietic and lymphoid tissues. IARC, Lyon2008: 54-63Google Scholar Based on the clinical presentation of the disease, cutaneous mastocytosis (CM) and systemic mastocytosis (SM) have been defined as the two main subtypes of mastocytosis.1Swerdlow S.H. Campo E. Harris N.L. Jaffe E.S. Pileri S.A. Stein H. Thiele J. Vardiman J.W. WHO classification of tumours of haematopoietic and lymphoid tissues. IARC, Lyon2008: 54-63Google Scholar CM primarily occurs in children, and the mast cell infiltration is confined to the skin.1Swerdlow S.H. Campo E. Harris N.L. Jaffe E.S. Pileri S.A. Stein H. Thiele J. Vardiman J.W. WHO classification of tumours of haematopoietic and lymphoid tissues. IARC, Lyon2008: 54-63Google Scholar In contrast, almost all adult patients with mastocytosis have SM, which is characterized by involvement of at least one extracutaneous organ and may involve multiple hematopoetic cell lineages.1Swerdlow S.H. Campo E. Harris N.L. Jaffe E.S. Pileri S.A. Stein H. Thiele J. Vardiman J.W. WHO classification of tumours of haematopoietic and lymphoid tissues. IARC, Lyon2008: 54-63Google Scholar, 2Metcalfe D.D. Mast cells and mastocytosis.Blood. 2008; 112: 946-956Crossref PubMed Scopus (417) Google Scholar In the vast majority of cases with SM, the clonal nature of the disease can be established through demonstration of a somatic A to T missense mutation at position 2447 of the coding sequence in the KIT gene.1Swerdlow S.H. Campo E. Harris N.L. Jaffe E.S. Pileri S.A. Stein H. Thiele J. Vardiman J.W. WHO classification of tumours of haematopoietic and lymphoid tissues. IARC, Lyon2008: 54-63Google Scholar, 3Garcia-Montero A.C. Jara-Acevedo M. Teodosio C. Sanchez M.L. Nunez R. Prados A. Aldanondo I. Sanchez L. Dominguez M. Botana L.M. Sanchez-Jimenez F. Sotlar K. Almeida J. Escribano L. Orfao A. KIT mutation in mast cells and other bone marrow hematopoietic cell lineages in systemic mast cell disorders: a prospective study of the Spanish Network on Mastocytosis (REMA) in a series of 113 patients.Blood. 2006; 108: 2366-2372Crossref PubMed Scopus (400) Google Scholar, 4Escribano L. Alvarez-Twose I. Sanchez-Munoz L. Garcia-Montero A. Nunez R. Almeida J. Jara-Acevedo M. Teodosio C. Garcia-Cosio M. Bellas C. Orfao A. Prognosis in adult indolent systemic mastocytosis: a long-term study of the Spanish Network on Mastocytosis in a series of 145 patients.J Allerg Clin Immunol. 2009; 124: 514-521Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar The resulting substitution of aspartate (D) to valine (V) at position 816 in the kinase domain leads to autoactivation of the KIT receptor tyrosine kinase.5Orfao A. Garcia-Montero A.C. Sanchez L. Escribano L. Recent advances in the understanding of mastocytosis: the role of KIT mutations.Br J Haematol. 2007; 138: 12-30Crossref PubMed Scopus (195) Google Scholar Studies with transgenic mice and mast cell lines have suggested that this mutation alone is sufficient to cause SM.6Ferrao P.T. Gonda T.J. Ashman L.K. Constitutively active mutant D816VKit induces megakayocyte and mast cell differentiation of early haemopoietic cells from murine foetal liver.Leuk Res. 2003; 27: 547-555Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 7Zappulla J.P. Dubreuil P. Desbois S. Letard S. Ben Hamouda N. Daeron M. Delsol G. Arock M. Liblau R.S. Mastocytosis in mice expressing human Kit receptor with the activating Asp816Val mutation.J Exp Med. 2005; 202: 1635-1641Crossref PubMed Scopus (76) Google Scholar, 8Mayerhofer M. Gleixner K.V. Hoelbl A. Florian S. Hoermann G. Aichberger K.J. Bilban M. Esterbauer H. Krauth M.T. Sperr W.R. Longley J.B. Kralovics R. Moriggl R. Zappulla J. Liblau R.S. Schwarzinger I. Sexl V. Sillaber C. Valent P. Unique effects of KIT D816V in BaF3 cells: induction of cluster formation, histamine synthesis, and early mast cell differentiation antigens.J Immunol. 2008; 180: 5466-5476Crossref PubMed Scopus (66) Google Scholar However, the KIT D816V mutation is not specific for mastocytosis, as gastrointestinal stromal tumors, acute myeloid leukemia, and germ cell tumors have also been demonstrated to carry this mutation.9Valent P. Akin C. Escribano L. Fodinger M. Hartmann K. Brockow K. Castells M. Sperr W.R. Kluin-Nelemans H.C. Hamdy N.A.T. Lortholary O. Robyn J. van Doormaal J. Sotlar K. Hauswirth A.W. Arock M. Hermine O. Hellmann A. Triggiani M. Niedoszytko M. Schwartz L.B. Orfao A. Horny H.P. Metcalfe D.D. Standards and standardization in mastocytosis: consensus statements on diagnostics, treatment recommendations and response criteria.Eur J Clin Invest. 2007; 37: 435-453Crossref PubMed Scopus (630) Google Scholar According to the 2008 World Health Organization classification of myeloproliferative neoplasms, the diagnosis of SM requires the presence of either the major criterion (≥15 mast cells in aggregates in an extracutaneous organ) and one of the four minor criteria (>25% mast cells with atypical morphology, codon 816 KIT mutation in an extracutaneous organ, mast cell CD2 and/or CD25 expression, and serum tryptase persistently >20 ng/ml) or three minor criteria.1Swerdlow S.H. Campo E. Harris N.L. Jaffe E.S. Pileri S.A. Stein H. Thiele J. Vardiman J.W. WHO classification of tumours of haematopoietic and lymphoid tissues. IARC, Lyon2008: 54-63Google Scholar In addition to diagnostic importance, the KIT D816V mutation is also important in the treatment of SM by causing resistance to Imatinib mesylate (Gleevec).10Pardanani A. Tefferi A. Systemic mastocytosis in adults: a review on prognosis and treatment based on 342 Mayo Clinic patients and current literature.Curr Opin Hematol. 2010; 17: 125-132Crossref PubMed Scopus (80) Google Scholar In contrast, the wild-type KIT and several rare KIT transmembrane (F522C) and juxtamembrane (V559G) mutations are reported to be Imatinib sensitive.11Akin C. Fumo G. Yavuz A.S. Lipsky P.E. Neckers L. Metcalfe D.D. A novel form of mastocytosis associated with a transmembrane c-kit mutation and response to imatinib.Blood. 2004; 103: 3222-3225Crossref PubMed Scopus (297) Google Scholar, 12Zermati Y. De Sepulveda P. Feger F. Letard S. Kersual J. Casteran N. Gorochov G. Dy M. Dumas A.R. Dorgham K. Parizot C. Bieche Y. Vidaud M. Lortholary O. Arock M. Hermine O. Dubreuil P. Effect of tyrosine kinase inhibitor STI571 on the kinase activity of wild-type and various mutated c-kit receptors found in mast cell neoplasms.Oncogene. 2003; 22: 660-664Crossref PubMed Scopus (163) Google Scholar Six different variants of SM have been defined: indolent SM (ISM), SM with an associated clonal hematological non-mast cell lineage disease (SM-AHNMD), aggressive SM (ASM), mast cell leukemia (MCL), mast cell sarcoma, and extracutaneous mastocytoma.1Swerdlow S.H. Campo E. Harris N.L. Jaffe E.S. Pileri S.A. Stein H. Thiele J. Vardiman J.W. WHO classification of tumours of haematopoietic and lymphoid tissues. IARC, Lyon2008: 54-63Google Scholar Patients with the indolent variant (ISM) have a life expectancy not significantly different from that of a sex- and age-matched population, but the median survival of SM-AHNMD, ASM, and MCL is significantly reduced.13Lim K.H. Tefferi A. Lasho T.L. Finke C. Patnaik M. Butterfield J.H. McClure R.F. Li C.Y. Pardanani A. Systemic mastocytosis in 342 consecutive adults: survival studies and prognostic factors.Blood. 2009; 113: 5727-5736Crossref PubMed Scopus (416) Google Scholar In a small fraction of patients with ISM, the disease will, however, progress into one of the more aggressive variants, and the presence of the KIT D816V mutation in non–mast cell lineages has recently been identified as one of the most powerful independent parameters for predicting this progression of ISM.4Escribano L. Alvarez-Twose I. Sanchez-Munoz L. Garcia-Montero A. Nunez R. Almeida J. Jara-Acevedo M. Teodosio C. Garcia-Cosio M. Bellas C. Orfao A. Prognosis in adult indolent systemic mastocytosis: a long-term study of the Spanish Network on Mastocytosis in a series of 145 patients.J Allerg Clin Immunol. 2009; 124: 514-521Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar The KIT D816V mutation allele burden has furthermore been proposed as a relevant and practical treatment endpoint in SM.14Pardanani A. Tefferi A. A critical reappraisal of treatment response criteria in systemic mastocytosis and a proposal for revisions.Eur J Haematol. 2010; 84: 371-378Crossref PubMed Scopus (23) Google Scholar At present, no single assay for KIT D816V mutation detection has been accepted as a general standard.14Pardanani A. Tefferi A. A critical reappraisal of treatment response criteria in systemic mastocytosis and a proposal for revisions.Eur J Haematol. 2010; 84: 371-378Crossref PubMed Scopus (23) Google Scholar Instead, three methodologies (RT-PCR + RFLP, PNA-mediated PCR, and allele-specific PCR) for mutation testing, applied with sufficient sensitivity, have been recommended.9Valent P. Akin C. Escribano L. Fodinger M. Hartmann K. Brockow K. Castells M. Sperr W.R. Kluin-Nelemans H.C. Hamdy N.A.T. Lortholary O. Robyn J. van Doormaal J. Sotlar K. Hauswirth A.W. Arock M. Hermine O. Hellmann A. Triggiani M. Niedoszytko M. Schwartz L.B. Orfao A. Horny H.P. Metcalfe D.D. Standards and standardization in mastocytosis: consensus statements on diagnostics, treatment recommendations and response criteria.Eur J Clin Invest. 2007; 37: 435-453Crossref PubMed Scopus (630) Google Scholar With the increasing focus on the clinical applications of the KIT D816V mutation in SM, there is a need for a molecular assay that allows quantification of low mutation levels. In the present study, we present an allele-specific real-time quantitative PCR (qPCR) assay that allows quantification of as little as 0.003% KIT D816V mutation-positive cells. We demonstrate the clinical relevance of the assay by identifying mutation-positive cell fractions as low as 0.03% in bone marrow aspirates from SM patients, and calculate the analytical sensitivity of samples that test mutation negative to determine the reliability of the result. Furthermore, we use the assay to identify SM patients with KIT D816V mutation in non-mast cells by comparing mutation-positive and mast cell fractions, and propose the assay for use in prospective studies of the KIT D816V allele burden as a treatment endpoint in SM. A total of 61 samples from 31 patients diagnosed with ISM (n = 18), ASM (n = 2), SM-AHNMD (n = 4), MCL (n = 1), and SM not classified to variant (n = 6), according to the 2008 World Health Organization classification of myeloproliferative neoplasms,1Swerdlow S.H. Campo E. Harris N.L. Jaffe E.S. Pileri S.A. Stein H. Thiele J. Vardiman J.W. WHO classification of tumours of haematopoietic and lymphoid tissues. IARC, Lyon2008: 54-63Google Scholar were analyzed for the presence of the KIT D816V mutation (Table 1). The patients were characterized to a varying degree because the major goal of the present study was to validate a new mutation detection method, and all available archival material from cases of SM was therefore included.Table 1Percentage KIT D816V Mutation–Positive Cells (qPCR % mut.) and Corresponding Assay Sensitivity (qPCR sens.)Case IDSexAge (years)Diagnosis (SM variant)Tissue typeSample date (month/day/year)qPCR % mut.qPCR sens.% Neoplastic mast cellsDNA sequencing1F38ISMPBMNC1/9/20060.040.02wtPBMNC9/9/20080.030.02wt2F48ISMPBMNC7/15/2009Neg.0.04wtBMMNC7/15/2009Neg.0.06Neg. (0.010)wt3M66ISMSkin biopsy6/4/2009157.5⁎Reduced analytical sensitivity in three skin biopsy samples with DNA extraction according to a previous protocol not including mechanical homogenization.wtPBMNC6/4/2009Neg.0.024M56ISMPBMNC9/10/20040.100.02wtPBMNC10/31/20070.170.02wt5M44ISMPBMNC7/5/20060.910.01wtPBMNC12/4/20074.80.01wt6M44ISMPBMNC10/28/20050.080.01wtBMMNC11/10/20050.690.05wtBMMNC6/4/20100.120.030.0207F59ISMPBMNC4/26/20070.420.02wt8M38ISMBMMNC2/9/20091.80.24wt9F57ISMPBMNC1/11/20070.410.03wtBMMNC2/2/20090.630.04wtBMMNC8/27/20090.100.00910F66ISMPBMNC9/23/20090.450.0211F66ISMSkin biopsy3/25/20103.10.19PBMNC4/9/20104.60.01BMMNC4/14/20104.90.020.15012F65ISMBMMNC1/13/20100.160.020.018PBMNC3/10/20100.060.0213M66ISMPBMNC5/31/20050.120.03wt14F50ISMSkin biopsy8/13/20092312⁎Reduced analytical sensitivity in three skin biopsy samples with DNA extraction according to a previous protocol not including mechanical homogenization.BMMNC9/4/20090.160.020.00815F60ISMPBMNC12/5/20070.060.02wtBMMNC6/26/20096.40.12wt16F61ISMPBMNC4/5/20059.10.01wtPBMNC9/1/2009430.04BMMNC9/4/2009970.020.10017M66ISMPBMNC4/21/2005Neg.0.04wtBMMNC7/27/20060.090.06wtPBMNC7/27/2006Neg.0.17wt18M58ISMBMMNC9/28/20060.030.01wtPBMNC9/28/2006Neg.0.05wt19F69ASMPBMNC3/21/2006240.0420F68ASMPBMNC8/5/20044.40.02wtPBMNC11/5/2007120.03wt21F80SM-AHNMD†BMMNC3/23/20104.00.01PBMNC4/7/20107.00.01Neg. (0.004)22M65SM-AHNMD‡PBMNC3/11/2010450.0123F71SM-AHNMD§PBMNC7/4/2008810.02Skin biopsy1/28/20108.20.0524M79SM-AHNMD¶PBMNC1/30/2006460.02D816V25M66MCLPBMNC7/5/2007Neg.0.04wt26F60Not classifiedPBMNC5/15/20081.10.02FFPE BM∥Aspiration clot.5/20/20083610D816VFFPE BM⁎⁎EDTA-decalcified biopsy sample.5/20/2008191.5wt27F46Not classifiedSkin biopsy7/20/20093.51.0⁎Reduced analytical sensitivity in three skin biopsy samples with DNA extraction according to a previous protocol not including mechanical homogenization.wtPBMNC2/9/20100.500.02BMMNC2/25/20100.640.0070.01828F48Not classifiedBMMNC11/25/20086.70.02wt29F53Not classifiedPBMNC9/15/20060.220.01wtPBMNC1/7/20100.460.02BMMNC1/7/20100.870.010.00830F44Not classifiedBMMNC12/9/2009220.010.220PBMNC1/22/201023Total leukocyte preparation using NH4Cl buffer for erythrocyte lysis of the same sample contained 37% mutation-positive cells. Likewise, PBMNC versus total leukocyte preparations from a mastocytosis patient not included in the present study contained 0.20% vs. 0.44% mutation-positive cells, respectively.0.02Neg. (0.006)31M51Not classifiedBMMNC4/7/20092.90.04wtPBMNC4/7/20090.690.02Neg. (0.010)M, male; F, female.The percentage KIT D816V mutation–positive cells (qPCR % mut.) and corresponding assay sensitivity (qPCR sens.) was measured in 61 samples from 31 cases of SM. In samples with no detectable mutation, results are expressed as mutation negative (neg.). A subset of samples were also analyzed for the presence of neoplastic mast cells using flow cytometry and KIT D816V mutation status using DNA sequencing (wt, wild-type, D816V, mutation detected). In samples with no detectable neoplastic mast cells (neg.), the sensitivity of the sample is indicated in parantheses.The AHNMD of the four cases of SM-AHNMD were as follows: JAK2 V617F mutation-positive essential thrombocythaemia (ET),† Myeloproliferative neoplasm, not otherwise specified (MPN, NOS),‡ (MPN, NOS),§ JAK2 V617F mutation-positive polycythaemia vera (PV).¶ Reduced analytical sensitivity in three skin biopsy samples with DNA extraction according to a previous protocol not including mechanical homogenization.∥ Aspiration clot. EDTA-decalcified biopsy sample.†† Total leukocyte preparation using NH4Cl buffer for erythrocyte lysis of the same sample contained 37% mutation-positive cells. Likewise, PBMNC versus total leukocyte preparations from a mastocytosis patient not included in the present study contained 0.20% vs. 0.44% mutation-positive cells, respectively. Open table in a new tab M, male; F, female. The percentage KIT D816V mutation–positive cells (qPCR % mut.) and corresponding assay sensitivity (qPCR sens.) was measured in 61 samples from 31 cases of SM. In samples with no detectable mutation, results are expressed as mutation negative (neg.). A subset of samples were also analyzed for the presence of neoplastic mast cells using flow cytometry and KIT D816V mutation status using DNA sequencing (wt, wild-type, D816V, mutation detected). In samples with no detectable neoplastic mast cells (neg.), the sensitivity of the sample is indicated in parantheses. The AHNMD of the four cases of SM-AHNMD were as follows: JAK2 V617F mutation-positive essential thrombocythaemia (ET),† Myeloproliferative neoplasm, not otherwise specified (MPN, NOS),‡ (MPN, NOS),§ JAK2 V617F mutation-positive polycythaemia vera (PV).¶ All patient samples were obtained from the diagnostic biobank at the Department of Pathology, Odense University Hospital, Denmark. The following types of samples were included in the study; mononuclear cells isolated using Ficoll-Paque density-gradient centrifugation from peripheral blood (PBMNCs; n = 35) and bone marrow aspirates (BMMNCs; n = 19); an EDTA-decalcified, FFPE bone marrow biopsy sample (n = 1); a FFPE bone marrow aspiration clot (n = 1); and skin biopsy samples that were snap-frozen in TissueTek OCT compound (n = 5). A patient sample previously identified to contain approximately 50% D816V mutation-positive KIT alleles using the Sanger dideoxy DNA sequencing technique was used as a positive control in all real-time qPCR experiments. Negative controls included non-mast cell leukemia cell lines (NB4 and K562) and PBMNCs from 20 healthy blood donors. All experiments were carried out in accordance with the Declaration of Helsinki and the Danish National Committee on Biomedical Research Ethics. DNA extraction was performed using the MagNA Pure LC Instrument (Roche Applied Science, Mannheim, Germany). DNA was extracted from cell lines, PBMNCs, and BMMNCs (106 cells) in a volume of 100 μL of elution buffer using the MagNA Pure LC DNA Isolation Kit I. DNA was extracted from FFPE bone marrow (10 μm section) in 200 μL of elution buffer using the MagNA Pure LC DNA Isolation Kit II, after deparaffinization with xylene and overnight proteinase K digestion. Skin biopsy samples ( 0.99. The cross-reaction with the wild-type allele of the mutation-specific assay was determined using genomic DNA from 20 healthy blood donor PBMNC samples and two non-mast cell leukemia cell lines (NB4 and K562). In all cases, the threshold cycle (Ct) value was at least 19 units higher in the mutation-specific assay compared with the control assay, corresponding to an assay sensitivity of 1/1.9419 = 0.0003% (Figure 1B). To be significant regarding detection of D816V mutated KIT alleles, a 10-fold higher cut-off limit, corresponding to 0.003%, was used to define positivity. Furthermore, a mutation-positive sample was defined to have sigmoidic amplification (log scale) and a Ct value of <44. The Ct value of 44 represents the Y-intercept Ct value of the standard curve, which corresponds to the Ct produced in a sample containing one D816V mutated KIT allele + one Ct, in line with the Europe Against Cancer guidelines.18Gabert J. Beillard E. van der Velden V.H.J. Bi W. Grimwade D. Pallisgaard N. Barbany G. Cazzaniga G. Cayuela J.M. Cave H. Pane F. Aerts J.L.E. De Micheli D. Thirion X. Pradel V. Gonzalez M. Viehmann S. Malec M. Saglio G. van Dongen J.J.M. Standardization and quality control studies of ‘real-time’ quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia—a Europe Against Cancer Program.Leukemia. 2003; 17: 2318-2357Crossref PubMed Scopus (1228) Google Scholar In accordance with the guidelines from the European Study Group on MRD in leukemia, “maximal reproducible sensitivity” was defined as the lowest dilution in which all replicates were positive within a 1.5 Ct range.19van der Velden V.H. Hochhaus A. Cazzaniga G. Szczepanski T. Gabert J. van Dongen J.J. Detection of minimal residual disease in hematologic malignancies by real-time quantitative PCR: principles, approaches, and laboratory aspects.Leukemia. 2003; 17: 1013-1034Crossref PubMed Scopus (445) Google Scholar Maximal reproducible sensitivity of the KIT D816V mutation-specific assay corresponded to Ct = 41. This Ct detection limit was therefore used to calculate the sensitivity of samples with limited amounts of DNA. Calculation of the KIT D816V mutation-positive cell fraction and assay sensitivity was performed using the following equation17Pfaffl M.W. A new mathematic
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