Clinical Chemistry 55:3
`587–598 (2009)
`
`Letters to the Editor
`
`Comparison of 7 Methods for
`Extracting Cell-Free DNA from
`Serum Samples of Colorectal
`Cancer Patients
`
`To the Editor:
`
`The presence of cell-free DNA of
`tumor origin in serum or plasma of
`cancer patients (1 ) has triggered
`numerous studies to explore the
`diagnostic and prognostic poten-
`tial of circulating DNA. This DNA,
`present only in minute concentra-
`tions in plasma or serum, is highly
`fragmented (2 ), a condition that
`often leads to substantial loss of
`DNA of small sizes in the course of
`DNA isolation. The lack of consen-
`sus
`regarding which extraction
`method is better for the efficient
`capture of such DNA might be par-
`tially responsible for the large dis-
`parities in the literature, which are
`reflected in reports of total concen-
`trations of plasma or serum DNA
`alone (3 ) or DNA integrity mea-
`surement as a diagnostic or prog-
`nostic tool. The ability to detect
`mutated v-Ki-ras2 Kirsten rat sar-
`coma viral oncogene homolog
`(KRAS) DNA in serum has been
`reported to vary with the chosen
`DNA isolation methods (4 ).
`We evaluated in parallel 7 iso-
`lation approaches (Table 1) by ex-
`tracting cell-free DNA from 12
`pooled sera obtained from 67 colo-
`rectal cancer patients and grouped
`on the basis of TNM tumor staging
`[tumor extent (T), spread to lymph
`nodes (N), and metastasis (M)].1
`The approaches we evaluated in-
`
`1 A total of 67 Chinese patients with sporadic colo-
`rectal cancers who underwent potentially curative
`surgical resection at the Department of Colorectal
`Surgery, Singapore General Hospital, between
`February 2004 and November 2005, participated
`in the present study. None of these patients had a
`known history of familial adenomatous polyposis,
`hereditary nonpolyposis colorectal cancer, or other
`types of cancers. None of them received preoper-
`ative chemotherapy or radiotherapy. The institu-
`tional review board of Singapore General Hospital
`approved this study, and all study participants
`provided written informed consent.
`
`volved diverse strategies for DNA
`isolation. The experimental set-up
`involved DNA isolation from a
`2-mL aliquot of serum in duplicate
`followed by DNA quantification by
`the fluorescent Quant-iT dsDNA
`HS assay (Invitrogen) and a Taq-
`man real-time PCR (rPCR) tech-
`nique on the cadherin 1, type 1, E-
`cadherin (epithelial) (CDH1) gene.
`To exclude false results in the
`CDH1 amplification due to various
`PCR inhibitors present in DNA ex-
`tracts, the isolated DNA was fur-
`ther quantified by Taqman rPCR
`on bisulfite-converted DNA be-
`cause the procedure of bisulfite-
`conversion of DNA removes many
`PCR-inhibitory components such
`as proteins, EDTA, and ethanol
`(EpiTech bisulfite kit, Qiagen). A
`fragment devoid of the CpG site of
`the actin, beta (ACTB) gene was
`the target for this round of rPCR.
`All 3 assays for DNA quantification
`were carried out in duplicate, and
`the DNA/gene amount in samples
`was interpolated by reference to
`corresponding
`standard curves
`generated by using 5 to 6 serial di-
`lutions of a DNA standard. Appro-
`priate blanks were included in each
`run. For rPCR, a positive control
`sample was run in each plate to
`control the interplate variation.
`The 7 extraction methods
`showed remarkable differences in
`the recovery of DNA from serum
`(Table 1). The phenol-chloroform
`procedure
`(PCI-glycogen),
`so-
`dium iodide method
`(NaI
`method),
`and QIAamp DNA
`blood kit generated significantly
`higher yields of DNA, assessed by
`fluorescent measurement, than the
`other 4 methods (all P ⬍ 0.05). As-
`sessed by rPCR targeting on CDH1
`(amplicon size: 68 bp), the NaI
`method was ranked top in the list,
`and statistical significance (P ⬍
`0.05) was achieved in all pairwise
`comparisons except with the PCI-
`glycogen approach. The latter,
`however, outperformed only the
`guanidine-resin procedure as de-
`
`the
`termined by application of
`Dunnett T3 test for pairwise multi-
`ple comparisons (Table 1). Because
`of the insufficient balance of DNA
`in the other 2 samples, we carried
`out the second round of rPCR on
`bisulfite-converted DNA in 10
`pooled samples. Among 3 methods
`evaluated, the NaI method exhib-
`ited the most abundant gene copy
`numbers of ACTB (amplicon size:
`115 bp), with the median of ACTB
`being 7 times higher than that gen-
`erated by the QIAamp DNA blood
`kit, although the difference did not
`reach the statistical significance
`(Table 1).
`The higher recovery of DNA
`obtained with the NaI and PCI gly-
`cogen procedures was also revealed
`on the agarose gel, which showed
`much stronger DNA signals along
`each of 2 lanes (electrophoretic im-
`age available on request). Interest-
`ingly, substantial amounts of small
`DNA fragments were recovered
`with these 2 methods, a result that
`was not achievable with the other 5
`protocols (Table 1). Furthermore,
`the size of these small fragments
`appeared to correspond to that of
`nucleosomal DNA, i.e., approxi-
`mately 180 –220 bp or its multiples.
`Because circulating DNA is highly
`fragmented, any isolation method
`that favors capture of fragmented
`DNA from serum or plasma will be
`useful for a variety of downstream
`applications in modern clinical
`and translational research labora-
`tories. These include efficient de-
`tection of mutations and capture of
`DNA methylation markers from
`serum or plasma.
`In comparing the PCI-glyco-
`gen approach and the NaI method,
`we found that the latter was not
`only superior to the former in
`terms of DNA quantity, as assessed
`by 2 rounds of rPCR, but also was
`simpler, more rapid, and less costly
`(data available on request).
`To summarize, the results of
`the present study, which involved 7
`isolation methods performed via
`
`587
`
`PGDX EX. 1017
`Page 1 of 12
`
`

`

`Letters to the Editor
`
`Table 1. Yield and fragment size of serum DNA isolated by 7 methods.
`
`DNA yield from 2 mL of serum
`
`By fluorescent
`assay, ng
`
`By rPCR on CDH1
`(relative amount to a
`positive control)
`
`By rPCR targeting
`ACTB on BSa
`templates
`(relative amount
`to a positive
`control)
`
`Visible bands on
`electrophoresis from 1 mL
`of serum
`
`Extraction method
`
`n
`
`Median (SE)
`
`n
`
`Median (SE)
`
`N Median (SE) ⬵200 bp ⬵400 bp ⬵500 bp
`
`PCI-glycogenb
`NaI methode
`Guanidine-resin methodg
`QIAamp DNA Blood Midi kit with
`carrier RNAh
`ChargeSwitch 1-mL serum kiti
`ZR serum DNA kitj
`Puregene DNA purification System
`Cell and Tissue Kitk
`
`12 367.958 (94.645)c 12 332.220 (119.031)d 10 15.507 (8.127)
`12 306.040 (61.228)c 12 391.735 (89.558)f
`10 17.476 (9.915)
`6
`8.928 (0.364)
`12
`1.145 (0.352)
`ND
`12 228.915 (38.162)c 12
`69.934 (14.869
`2.247 (2.601
`
`10
`
`12
`6
`12
`
`83.165 (13.370)
`15.363 (6.580)
`59.200 (11.652)
`
`12
`12
`12
`
`74.978 (16.612)
`6.577 (5.749)
`42.094 (19.447)
`
`ND
`ND
`
`Yes
`Yes
`No
`No
`
`No
`No
`No
`
`Yes
`Yes
`No
`No
`
`No
`No
`No
`
`Yes
`Yes
`Yes
`Yes
`
`Yes
`Yes
`Yes
`
`a BS, bisulfite-converted; ND, not detected.
`b Phenol-chloroform method with addition of glycogen.
`c DNA yield was significantly higher than that isolated by the methods without any mark in the column (P ⬍ 0.05, by Friedman analysis followed by Dunnett T3
`test for pairwise multiple comparisons), but there was no difference among marked methods.
`d CDH1 quantity was second highest among all 7 methods but statistical significance was reached only in pairwise comparison with the guanidine-resin method
`(P ⬍ 0.05).
`e Sodium iodide method (protocol available on request).
`f CDH1 quantity was highest among all 7 methods and of statistical significance in all pairwise comparisons except with the PCI-glycogen procedure (P ⬍ 0.05).
`g Adapted from the method described by Wang et al. (4).
`h Performed according to manufacturer’s instructions except with addition of 3 ␮g of carrier RNA into each sample; catalog no. 51183, lot no. 127136152.
`i Performed according to manufacturer’s instructions, catalog no. CS 11040, lot no. 1409017.
`j Performed according to manufacturer’s instructions, catalog no. D3013, lot no. 32-200607019.
`k Performed according to manufacturer’s instructions, part no. D-5500A, lot no. GS18781.
`
`quantification of serum DNA by 3
`assays and examination of frag-
`ment sizes of DNA isolated by elec-
`trophoresis, indicated that the NaI
`procedure consistently revealed
`better performance. Therefore,
`this procedure appears to be a suit-
`able method for cell-free DNA ex-
`traction for many downstream ap-
`plications in cancer research.
`
`Author Contributions: All authors confirmed
`they have contributed to the intellectual content of
`this paper and have met the following 3 require-
`ments: (a) significant contributions to the concep-
`tionanddesign,acquisitionofdataoranalysis,and
`interpretation of data; (b) drafting or revising the
`article for intellectual content; and (c) final ap-
`proval of the published article.
`
`Authors’ Disclosures of Potential Conflicts of
`Interest: Upon manuscript submission, all au-
`thors completed the Disclosures of Potential Con-
`flict of Interest form. Potential conflicts of interest:
`
`588 Clinical Chemistry 55:3 (2009)
`
`Employment or Leadership: S.L. Fong, Singa-
`pore General Hospital; K.W. Eu, Singapore
`General Hospital; Y. Liu, Singapore General
`Hospital.
`Consultant or Advisory Role: None de-
`clared.
`Stock Ownership: None declared.
`Honoraria: None declared.
`Research Funding: Grant from the Na-
`tional Medical Research Council (NMRC),
`Singapore.
`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, preparation or approval of manuscript.
`
`Acknowledgments: We would thank Bing
`Zhou Li (Research Operation, Duke-NUS
`Graduate Medical School, Singapore) for
`his assistance in the quantification of DNA.
`
`References
`
`1. Anker P, Mulcahy H, Chen Xq, Stroun M. Detec-
`tion of circulating tumour DNA in the blood
`
`(plasma/serum) of cancer patients. Cancer Me-
`tastasis Rev 1999;18:65–73.
`2. Jahr S, Hentze H, Englisch S, Hardt D, Fackelmayer FO,
`Hesch RD, Knippers R. DNA fragments in the blood
`plasma of cancer patients: quantitations and evidence
`for their origin from apoptotic and necrotic cells. Can-
`cer Res 2001;61:1659–65.
`3. Boddy JL, Gal S, Malone PR, Harris AL, Wain-
`scoat JS. Prospective study of quantitation of
`plasma DNA levels in the diagnosis of malignant
`versus benign prostate disease. Clin Cancer Res
`2005;11:1394 –9.
`4. Wang M, Block TM, Steel L, Brenner DE, Su YH.
`Preferential
`isolation of fragmented DNA en-
`hances the detection of circulation mutated K-
`ras DNA. Clin Chem 2004;50:211–3.
`
`Siew Lee Fong2
`Ji Tuan Zhang3
`Che Kang Lim4
`Kong Weng Eu2
`Yanqun Liu2*
`
`2 Department of Colorectal Surgery
`Singapore General Hospital
`Singapore
`
`PGDX EX. 1017
`Page 2 of 12
`
`

`

`Letters to the Editor
`
`3 Department of Orthopedic Surgery
`Yong Loo Lin School of Medicine
`National University of Singapore
`Singapore
`4 Department of Clinical Research
`Singapore General Hospital
`Singapore
`
`* Address correspondence to this author at:
`Department of Colorectal Surgery
`Singapore General Hospital
`Outram Road
`Singapore 169608
`Fax 65-62262009
`E-mail liu.yan.qun@sgh.com.sg
`
`DOI: 10.1373/clinchem.2008.110122
`
`Cross-Reactivity of
`Phentermine with an
`Immunoassay Designed to
`Detect Amphetamine in a
`Meconium Specimen
`
`To the Editor:
`
`Neonates exposed to drugs of abuse
`in utero can experience prenatal
`drug dependence leading to with-
`drawal symptoms and a number of
`other health problems (1). Early de-
`tection of exposure is critical to guide
`necessary treatment and improve
`outcomes for these children. Meco-
`nium begins to form in the digestive
`tract at 12–16 weeks gestation. Drugs
`and metabolites collect in meco-
`nium beginning at about 5 months
`gestation. Thus, meconium testing
`can identify exposure to drugs dur-
`ing the last 4 months of a full-term
`pregnancy (2).
`Our
`laboratory uses ELISA
`reagents (Immunalysis) to detect
`drugs of abuse in meconium. Poor
`specificity of immunoassay reagents
`for amphetamines is well character-
`ized and as a result, specimens that
`test positive for amphetamines by
`immunoassay are routinely tested
`by a second analytical method to
`prevent false-positive results. Our
`ELISA screen for meconium has sep-
`
`arate detection antibodies for am-
`phetamine and methamphetamine.
`The ELISA cutoff for these drugs is
`20 ng/g. All positive screen results are
`confirmed by GC-MS.
`We report the investigation of
`an unconfirmed positive amphet-
`amine result. ELISA assay of the
`meconium specimen in question
`was positive for amphetamine but
`negative for methamphetamine. The
`confirmation assay failed to detect
`amphetamine, methamphetamine,
`3,4-methylenedioxyamphetamine,
`or 3,4-methylenedioxy-N-methyl-
`amphetamine. The specimen was
`then analyzed with a more compre-
`hensive
`liquid-chromatography–
`tandem mass-spectrometry method
`to scan for possible candidates that
`could elicit a positive screen result.
`Briefly, 0.25 g of meconium was
`extracted with 1 mL of 25 mmol/L
`phosphate buffer, pH 7.3, and cen-
`trifuged. Solid-phase extraction on a
`250-␮L aliquot was perfomed with a
`mixed-mode column. Deuterated
`analogs were added as internal stan-
`dards. The drugs were eluted with 2
`mL of methanol: ammonium hy-
`droxide (98:2 vol:vol), evaporated to
`dryness under nitrogen, and recon-
`stituted in 50 ␮L methanol. Analyses
`were performed with an Agilent
`Technologies 1200 series
`liquid
`chromatograph coupled to a 6410
`triple quadrupole mass spectrome-
`ter operated in the positive electro-
`spray mode and equipped with a
`Zorbax Eclipse XDB C18 column
`(4.6 ⫻ 50 mm, 1.8 ␮m) at 40 °C and
`an
`ammonium-acetate:methanol
`mobile phase. The specimen was
`found to contain 191 ng/g phenter-
`mine, an anorexiant stimulant rec-
`ommended for short-term use in the
`treatment of obesity.
`We quantified phentermine by
`using d5-methamphetamine as the
`internal standard. Because the mo-
`lecular weight of methamphetamine
`and phentermine are the same, the
`transitions for liquid chromatogra-
`phy–tandem mass
`spectrometry
`were also the same, so the com-
`
`pounds were separated on the basis
`of retention time (methamphet-
`amine 5.9 min; phentermine 6.6
`min). Two transitions were selected:
`The quantifying transition was m/z
`150.2–91.1 and the qualifying transi-
`tion was m/z 150.2–65.1. Ion ratios
`were within ⫾20% to meet the crite-
`rion for a positive result. The moni-
`tored transition for the d5-meth-
`amphetamine was m/z 155.2–92.1.
`The ELISA kit insert indicates
`that phentermine has 89% cross-re-
`activity at 25 ng/g with the amphet-
`amine antibody, but does not cross-
`react with the methamphetamine
`antibody. Phentermine was fortified
`at 3 concentrations (100, 200, and
`400 ng/g) and run as unknowns in
`the amphetamine and methamphet-
`amine ELISA. The phentermine-
`fortified samples showed changes in
`binding (B/B0) of 44%, 29%, and
`16% in the amphetamine assay,
`whereas the methamphetamine as-
`say showed negligible change in
`binding (B/B0) at 88%, 86%, and
`85%. B/B0 is defined as the absor-
`bance reading of the sample (B) di-
`vided by the absorbance reading of
`the zero-dose standard (B0). The
`change in B/B0 for phentermine ob-
`tained by using both kits is shown in
`Fig. 1. Although phentermine and
`methamphetamine have the same
`molecular weight of 149 Da, they dif-
`fer in the position of the additional
`methyl group. Therefore, coupling
`of the antibody through the aro-
`matic ring of amphetamine, rather
`than the nitrogen, minimizes cross-
`reactivity with methamphetamine
`but still allows cross-reactivity with
`the amphetamine antibody.
`To the best of our knowledge,
`this is the only reported incidence of
`the confirmation and quantification
`of phentermine in a meconium
`specimen. Phentermine is a preg-
`nancy category C drug, indicating it
`should not be administered during
`pregnancy unless clearly needed be-
`cause no data are available to deter-
`mine if it adversely affects the fetus.
`Although no evidence of toxicity or
`
`Clinical Chemistry 55:3 (2009) 589
`
`PGDX EX. 1017
`Page 3 of 12
`
`

`

`Letters to the Editor
`
`Fig. 1. Change in binding (B/B0) of phentermine to amphetamine and
`methamphetamine ELISA antibody.
`
`lethality was observed in rat pups
`from administering of phentermine
`to dams (3), 5 adverse outcomes
`(stillbirths) occurred in a previous
`study of 118 pregnant women taking
`phentermine (4). This case demon-
`strates the importance of performing
`confirmatory testing for drugs of
`abuse and supports a need to include
`phentermine in confirmatory testing
`to distinguish its use or abuse from
`that of amphetamine or other re-
`lated drugs.
`
`Author Contributions: All authors con-
`firmed they have contributed to the intellec-
`tual content of this paper and have met the
`following 3 requirements: (a) significant con-
`tributions to the conception and design, ac-
`quisition of data or analysis, and interpreta-
`tion of data; (b) drafting or revising the article
`for intellectual content; and (c) final approval
`of the published article.
`
`Authors’ Disclosures of Potential Con-
`flicts of Interest: Upon manuscript submis-
`sion, all authors completed the Disclosures of
`Potential Conflict of Interest form. Potential
`conflicts of interest:
`
`Employment or Leadership: C. Moore,
`Immunalysis Corporation.
`Consultant or Advisory Role: None de-
`clared.
`Stock Ownership: None declared.
`Honoraria: None declared.
`Research Funding: None declared.
`Expert Testimony: None declared.
`
`Role of Sponsor: The funding organiza-
`tions played no role in the design of study,
`choice of enrolled patients, review and in-
`terpretation of data, preparation or ap-
`proval of manuscript.
`
`590 Clinical Chemistry 55:3 (2009)
`
`References
`
`1. Kwong TC, Ryan RM. Detection of intrauterine
`illicit drug exposure by newborn drug testing.
`Clin Chem 1997;43:235– 42.
`2. Gareri J, Klein J, Koren G. Drugs of abuse testing
`in meconium. Clin Chim Acta 2006;366:101–11.
`3. Thoma-Laurie D, Walker ER, Reasor MJ. Neonatal
`toxicity in rats following in utero exposure to chlor-
`phentermine or phentermine. Toxicology 1982;24:
`85–94.
`4. Sands RX. Obesity and pregnancy: weight con-
`trol with a resinate. Am J Obstet Gynecol 1962;
`83:1617–21.
`
`Stephanie J. Marin1*
`Christine Moore2
`Gwendolyn A. McMillin3,4
`
`1 ARUP Institute for Clinical and
`Experimental Pathology
`Salt Lake City, UT
`2 Immunalysis Corporation
`Pomona, CA
`3 Department of Pathology
`University of Utah School of Medicine
`Salt Lake City, UT
`4 ARUP Laboratories
`Salt Lake City, UT
`
`* Address correspondence to this author at:
`ARUP Institute for Clinical and
`Experimental Pathology
`500 Chipeta Way
`Salt Lake City, UT 84108
`Fax 011-801-584-5048
`E-mail stephanie.marin@aruplab.com
`
`DOI: 10.1373/clinchem.2008.114603
`
`New Highly Sensitivity Assay
`Used to Measure Cardiac
`Troponin T Concentration
`Changes During a Continuous
`216-km Marathon
`
`To the Editor:
`
`We recently measured cardiac tro-
`ponin T (cTnT)1 concentrations
`serially in blood from 10 partici-
`pants of the Badwater ultramara-
`thon, a continuous 216-km race
`that takes place under extreme en-
`vironmental conditions. Details on
`the Badwater ultramarathon itself,
`and on the training experience and
`health condition of the athletes
`have been reported previously (1 ).
`Briefly, the athletes had completed
`a mean of 43.4 (median 20, range
`5–130) marathons and 20.4 (me-
`dian 15, range 6 – 80) ultramara-
`thons, and the mean finishing
`times for the Badwater race were
`51.24 h (median 45.2 h, range
`43.5– 61.4 h). cTnT was measured
`with a new highly sensitive assay
`for cardiac troponin T (Hs-TnT)
`(Roche Diagnostics) on an ELECSYS
`2010 automated analyzer that uses
`chemiluminescence technology.
`As described previously (2 ), the in-
`terassay CV of this assay is 8% at 10
`ng/L and 2.5% at 100 ng/L, and the
`intraassay CV is 5% at 10 ng/L and
`1% at 100 ng/L. The diagnostic
`range of this assay is 2 to 10000
`ng/L.
`Blood samples (EDTA plasma)
`were drawn at baseline, after the first
`half marathon, after each full mara-
`thon distance, and at the finish. In a
`previous study we tested the third-
`generation cTnT assay in the same
`study population and were unable to
`detect cTnT concentrations above
`the lower limit of detection in any
`blood sample before or during the
`
`1 Nonstandard abbreviations: cTnT, cardiac troponin
`T; Hs-TnT, highly sensitive assay for cardiac tro-
`ponin T; BNP, brain natriuretic peptide.
`
`PGDX EX. 1017
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`
`

`

`Letters to the Editor
`
`peptide (BNP). In animal models,
`cytoprotective and growth-regulating
`effects of BNP have been reported
`(5). BNP has been shown to open
`adenosine-triphosphate–sensitive
`potassium channels of myocardial
`mitochondria via the natriuretic
`peptide receptor A signaling path-
`way. Furthermore, BNP exerts
`counterregulatory and sympathoin-
`hibitory effects inhibiting hypertro-
`phy in cultured cardiac myocytes
`and angiotensin-II–stimulated col-
`lagen synthesis by cardiac fibro-
`blasts. Supporting this hypothesis, in
`our study population N-terminal
`pro-BNP, the biologically inactive
`cleavage product of pro-BNP, in-
`creased in 9 of 10 runners (inade-
`quate sampling in 1 case), and peak
`values of N-terminal pro-BNP were
`significantly higher (424 vs 126 ng/L,
`P ⫽ 0.0063) in those ultraendurance
`runners who demonstrated an in-
`crease of cardiac troponin above the
`99th percentile value as measured
`with the new more sensitive and pre-
`cise troponin T assay (Fig. 1B). Al-
`though a potential limitation of this
`study is that blood sampling was not
`complete in all cases, a consistent
`change supported by 2 sequential
`samples was present for the most im-
`portant observations. Assessment of
`right and left ventricular perfor-
`mance and hemodynamics would
`have been informative but was not
`available in this cohort. Owing to the
`small number of individuals studied
`and the heterogeneity of the release
`patterns, confirmatory follow-up
`studies are needed. Nevertheless, the
`observation is interesting and has al-
`lowed generation of a hypothesis
`that should be validated in future
`studies.
`
`Author Contributions: All authors con-
`firmed they have contributed to the intellec-
`tual content of this paper and have met the
`following 3 requirements: (a) significant con-
`tributions to the conception and design, ac-
`quisition of data, or analysis and interpreta-
`tion of data; (b) drafting or revising the article
`
`Clinical Chemistry 55:3 (2009) 591
`
`Fig. 1. Concentrations of Hs-TnT (A) and N-terminal pro-BNP (NT-proBNP)
`(B) in individual runners during a continuous 216-km ultramarathon.
`E0 to E8 denote the time points of blood sampling: E0, half-marathon distance;
`E1, full-marathon distance; E3, double-marathon distance; E4, quadruple-mara-
`thon distance; E7, finish; E8, shortly after E7.
`
`ultramarathon (1). We now report
`that with the new Hs-TnT assay,
`cTnT was measurable in all samples
`including the baseline samples. The
`time course of individual Hs-TnT
`values is displayed in Fig. 1A. We ob-
`served several patterns of cTnT con-
`centration changes detected with
`over time with the Hs-TnT assay, in-
`cluding cases with constantly low
`concentrations without changes as
`well as cases with an rise of Hs-TnT
`early after the start of strenuous ex-
`ercise. In one case, Hs-TnT de-
`creased to a low preexercise concen-
`tration while the athlete was still
`running. The latter case is extremely
`
`interesting because this observation
`suggests a physiological counter-
`regulatory process rather than a sim-
`ple increase of myocardial damage
`related to the intensity of exercise. It
`is tempting to speculate that such an
`adaptation mechanism could be
`meaningful in limiting the magni-
`tude of myonecrosis. Previous stud-
`ies on endurance athletes suggested
`that training intensity and the type of
`exercise were major determinants of
`the rate and magnitude of subse-
`quent
`troponin release (3, 4). A
`mechanism that in our opinion may
`contribute to this adaptation mech-
`anism is release of brain natriuretic
`
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`

`

`Letters to the Editor
`
`for intellectual content; and (c) final approval
`of the published article.
`
`Authors’ Disclosures of Potential Con-
`flicts of Interest: Upon manuscript submis-
`sion, 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: E. Giannitsis, Merck Sharp &
`Dohme, Lilly, Bristol Myers Squibb, Glaxo-
`SmithKline, Roche Diagnostics, and Bayer;
`H.J. Roth, Merck Sharp & Dohme and
`Roche Diagnostics.
`Research Funding: E. Giannitsis received
`funds and access to assays from Roche Diag-
`nostics.
`Expert Testimony: None declared.
`
`Role of Sponsor: The funding organiza-
`tions played no role in the design of study,
`choice of enrolled patients, review and in-
`terpretation of data, preparation or ap-
`proval of manuscript.
`
`References
`
`1. Roth HJ, Leitha¨ user RM, Doppelmayr H, Doppel-
`mayr M, Finkernagel H, von Duvillard SP, et al.
`Cardiospecificity of the 3rd generation cardiac
`troponin T assay during and after a 216 km
`ultra-endurance marathon run in Death Valley.
`Clin Res Cardiol 2007;96:359 – 64.
`2. Kurz K, Giannitsis E, Zehelein J, Katus HA. Highly
`sensitive cardiac troponin T values remain con-
`stant after brief exercise- or pharmacologic-
`induced reversible myocardial
`ischemia. Clin
`Chem 2008;54:1234 – 8.
`3. Shave R, George KP, Atkinson G, Hart E, Middle-
`ton N, Whyte G, et al. Exercise-induced cardiac
`troponin T release: a meta-analysis. Med Sci
`Sports Exerc 2007;39:2099 –106.
`4. Neilan TG,
`Januzzi
`JL, Lee-Lewandrowski E,
`Ton-Nu TT, Yoerger DM, Jassal DS, et al. Myo-
`cardial injury and ventricular dysfunction related
`to training levels among nonelite participants in
`the Boston marathon. Circulation 2006;114:
`2325–33.
`5. Brunner-La Rocca HP, Kaye DM, Woods RL, Hast-
`ings J, Esler MD. Effects of intravenous brain
`natriuretic peptide on regional sympathetic ac-
`tivity in patients with chronic heart failure as
`compared with healthy control subjects. J Am
`Coll Cardiol 2001;37:1221–7.
`
`2 Medizinische Klinik, Abteilung fu¨r Innere
`Medizin III, Universita¨tsklinikum
`Heidelberg, Germany
`3 Laboratory Limbach and Colleagues
`Heidelberg, Germany
`4 Centre for Sports and Exercise Science
`Department of Biological Sciences
`University of Essex
`Colchester, England
`5 Department of Sports Medicine
`University Potsdam
`Potsdam, Germany
`
`* Address correspondence to this author at:
`University of Heidelberg
`Abteilung Innere Medizin III
`Bergheimer Strasse 58
`Heidelberg, Germany 69115
`Fax ⫹49-6221-5516
`E-mail evangelos_giannitsis@med.uni-
`heidelberg.de
`
`DOI: 10.1373/clinchem.2008.116566
`
`AQP4-IgG
`Immunoprecipitation Assay
`Optimization
`
`To the Editor:
`
`The sensitivity and specificity of
`tissue-based immunofluorescence
`assays (IFA)1 is established for de-
`tecting neuromyelitis optica (NMO)
`IgG as an aid to classifying inflam-
`matory demyelinating central ner-
`vous system disorders belonging
`to the NMO spectrum (1 ): NMO,
`partial and inaugural
`forms of
`NMO [longitudinally extensive
`transverse myelitis (ⱖ3 vertebral
`segments radiologically) and re-
`current optic neuritis] and pediat-
`ric
`inflammatory
`autoimmune
`encephalopathies.
`Immunopre-
`cipitation assays (IPA) employing
`
`Evangelos Giannitsis2*
`Heinz Juergen Roth3
`Renate M. Leitha¨user4
`Juergen Scherhag5
`Ralt Beneke4
`Hugo A. Katus2
`
`1 Nonstandard abbreviations: IFA, immunofluores-
`cence assay; NMO, neuromyelitis optica;
`IPA,
`immunoprecipitation
`assays;
`AQP4,
`human
`aquaporin-4; GFP, green fluorescent protein;
`MuSK, muscle-specific kinase;
`IRB,
`Institutional
`Review Board.
`
`592 Clinical Chemistry 55:3 (2009)
`
`recombinant human aquaporin-4
`(AQP4), the antigen of NMO, have
`been described (2, 3 ). In optimiz-
`ing the green fluorescent protein
`(GFP)-tagged AQP4 IPA (2 ) for
`high-throughput testing, we en-
`countered cases in which patient
`IgG bound to GFP and not to
`AQP4. Here we report our efforts,
`in a clinical setting, to determine
`the rate of false positivity and take
`steps to eliminate it.
`From October 1, 2007 to
`March 31, 2008, the Mayo Clinic
`Neuroimmunology
`Laboratory
`evaluated, by use of GFP-AQP4
`IPA, sera from 117 healthy adult
`control individuals (group 1) and
`from 5500 patients for whom ser-
`vice NMO-IgG IFA testing was re-
`quested. Of these, 557 were IFA-
`positive (group 2) and 4943 were
`IFA negative (group 3). We also
`tested neurologically asymptom-
`atic patients, 58 with hypergam-
`maglobulinemia (group 4) and 27
`with, systemic lupus erythemato-
`sus or Sjo¨gren syndrome (group 5).
`In the course of clinical sero-
`logical evaluation for NMO-IgG
`(indirect immunofluorescence as-
`say performed on a service basis
`in the Mayo Clinic Department
`of Laboratory Medicine and Pa-
`thology), patients were identified.
`Medical records were reviewed
`retrospectively. The Institutional
`Review Board (IRB) of the Mayo
`Clinic College of Medicine, Roch-
`ester, MN, approved the study
`(IRB 07-007453).
`We added 30 ␮L of serum to
`duplicate 85-␮L aliquots of recom-
`binant human GFP-AQP4 [150 000
`GFP counts, solubilized in lysis
`buffer (700 mmol/L NaCl, 10 mmol/L
`Tris-HCl pH 8.0, 1 mmol/L EDTA,
`0.5% Triton X-100)] from human
`embryonic kidney–293 cells trans-
`fected with pEGFP-AQP4 (plasmid-
`DNA–encoding enhanced GFP-
`AQP4) (2) and held 16 h at 4 °C.
`Control specimens contained 4 dif-
`ferent pools of normal human se-
`rum samples or 1 serum sample
`
`PGDX EX. 1017
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`

`Letters to the Editor
`
`immunoprecipitation of GFP must
`be considered when sera are evalu-
`ated by using recombinant auto-
`antigens fused with this tag. This
`consideration is of particular im-
`portance in patients who are sero-
`negative for NMO-IgG by stan-
`dardized IFA (group 3 in our
`study). Among 80 such patients,
`we obtained a false-positive rate of
`5%. After we eliminated those pa-
`tients from consideration, all but
`1 of the remaining 16 patients
`who had serum samples positive by
`IPA alone and for whom clinical
`information was available were
`confirmed to have a clinical disor-
`der recognized to be in the NMO
`spectrum. The exceptional sero-
`positive patient had a diagnosis of
`monophasic optic neuritis. One
`can reasonably speculate that se-
`ropositivity in this case predicts
`future relapse. False-positive re-
`sults analogous to those we report
`for the GFP-AQP4 assay have
`been reported in immunoprecipi-
`tation assays for the muscle nico-
`tinic acetylcholine receptor anti-
`body (125I-␣-bungarotoxin tag) (4)
`and muscle-specific kinase (MuSK)
`antibody (placental alkaline phos-
`phatase tag) (5 ). The results of this
`large study suggest that our opti-
`mized high-throughput IPA using
`GFP-AQP4 is not as sensitive as the
`originally described tissue-based
`IFA for detecting NMO-IgG. How-
`ever, as an adjunct to IFA, the IPA
`described here enhances the diag-
`nostic yield of NMO-spectrum dis-
`orders. A more formal comparison
`of sensitivity and specificity of IFA,
`IPA, and combined assays in con-
`secutively-acquired patients with
`clinical
`information available in
`each case is underway. Assurance
`of assay specificity requires elimi-
`nation of false-positive results to
`preclude misdiagnosis and inap-
`propriate long-term commitment
`of patients to immunotherapies
`with attendant expense and risks.
`
`Clinical Chemistry 55:3 (2009) 593
`
`Fig. 1. Frequency of false-positive AQP4 immunoprecipitation in 20 patients
`(group 3) who were negative for NMO-IgG by IFA but positive by GFP-AQP4
`immunoprecipitation assay, and for whom clinical information was available.
`Subtraction of values for GFP alone eliminated 4 patients (false positives, 5%)
`with diagnoses: 䡺, NMO-spectrum disorder; ‚, single-episode optic neuritis; F,
`(corrected values negative) neuroretinitis, primary progressive multiple sclerosis,
`neurosarcoidosis, or paraneoplastic optic neuropathy.
`
`from an AQP4-IgG–positive patient.
`Two hours after adding protein-G
`sepharose 4B (30 ␮L; Zymed Labo-
`ratories), we washed the beads 6
`times with lysis buffer and measured
`GFP spectrophotometrically (exci-
`tation, 485 nm; emission, 535 nm).
`After
`subtracting the maximum
`value yielded by normal control sera,
`we calculated GFP-AQP4 bound
`(nmol/L serum) by reference to a
`GFP standard (Clontech). Sera
`yielding values ⬎10 nmol/L serum
`were clarified by centrifugation and
`reassayed with GFP-AQP4 and with
`GFP alone (150 000 counts). The fi-
`nal results represent each patient’s
`mean GFP-AQP4 value after sub-
`tracting the individual’s value for
`precipitation of GFP alone.
`Analysis of serum from groups
`1, 4 and 5 yielded a final GFP-
`AQP4 concentration ⱕ10 nmol/L
`serum. In group 2, of 557 sera pos-
`itive by IFA, none precipitated
`GFP alone, and 331 yielded initial
`and finally corrected AQP4-IgG
`concentrations ⬎10 nmol/L (59%;
`median 46.6, range 10.1–3661 nmol/
`L). Clinical information was avail-
`
`able for 54 of these 331 patients
`(16%). All had an NMO-spectrum
`disorder: 28 patients had NMO, 24
`had longitudinally extensive trans-
`verse myelitis (recurrent in 12 pa-
`tients), and 2 had recurrent optic
`neuritis. In group 3, of 49