pected, given that the two assays weidentified as susceptible to parapro-tein interference are used by 396(10%) and 213 (5%), respectively, ofthe 3971 laboratories participating inthe College of American Pathologists2003 C-B HDL-C survey (7 ).

References1. Levy Y, Aviram M, Spira G, Tatarsky I, Brook GJ,

Carter A. Plasma cholesterol concentration andextra lipid band in monoclonal gammopathies.Postgrad Med J 1984;60:44953.

2. Kadri N, Douville P, Lachance P. Monoclonalparaprotein may interfere with the Roche DirectHDL-C Plus Assay. Clin Chem 2002;48:964.

3. C-C chemistry participant summary report, sur-veys 2002. Northfield, IL: College of AmericanPathologists, 2002.

4. Abel G, Laposata M. Lipids, lipoproteins andcardiovascular risk assessment. In: Lewand-rowski K, ed. Clinical chemistry. Laboratory man-agement & clinical correlations. Philadelphia:Lippincott Williams & Wilkins, 2002:57591.

5. Hachem H, Favre G, Ghalim N, Puchois P, Fru-chart JC, Soula G. Quantitative abnormalities oflipoprotein particles in multiple myeloma. J ClinChem Clin Biochem 1987;25:6759.

6. Hachem H, Favre G, Soula G. Evidence forqualitative abnormalities in high-density lipopro-teins from myeloma patients: the presence for

amyloid A protein could explain HDL modifica-tions. Biochim Biophys Acta 1988;25:2717.

7. C-B chemistry participant summary report, sur-veys 2003. Northfield, IL: College of AmericanPathologists, 2003.

Arthur Baca1

Richard J. Haber2

Kirk Sujishi3

Philip H. Frost2

Valerie L. Ng1*

Departments of 1 Laboratory Medicineand 2 Medicine

University of California San FranciscoSan Francisco, CA 94110

3 Clinical LaboratoryUniversity of California San Francisco

Medical CenterSan Francisco, CA 94110

*Author for correspondence.

DOI: 10.1373/clinchem.2003.027813

EDTA Is a Better Anticoagulant thanHeparin or Citrate for Delayed BloodProcessing for Plasma DNA Analysis

To the Editor:Plasma is usually chosen for cell-freecirculating DNA studies becauseex vivo release of DNA from hema-topoietic cells was demonstrated inserum during the clotting processfrom whole blood (13). Among themost commonly used anticoagulants,EDTA, heparin, and citrate, heparinwas suggested by some investigatorsto be an unsuitable anticoagulant be-cause it could inhibit PCR (46).However, the quantitative effects ofthe choice of anticoagulants forplasma DNA analysis have not beenstudied systematically. We thereforeconducted an experiment to investi-gate the effect of three commonlyused anticoagulants on plasma DNAquantification.

Ten healthy individuals were re-cruited, and peripheral blood wascollected into four EDTA, four hepa-rin, and four citrate tubes (Vacuette;Greiner) for each individual. Bloodsamples were kept at room tempera-ture (24 C). Plasma was obtained bycentrifugation from the EDTA, hepa-rin, and citrate tubes at four differenttime points, 0, 2, 6, and 24 h aftervenesection, respectively, with onetube used for each time point. Theplasma samples were stored at80 C until analysis. DNA was ex-tracted from 200 L of plasma ac-cording to the blood and body fluidprotocol of the QIAamp Blood Kit(Qiagen), with a final elution volumeof 50 L. We then subjected 5 L ofthe DNA to real-time quantitativePCR for the -globin gene as de-scribed previously (7 ) on a 7700Sequence Detector (Applied Biosys-tems). DNA concentration is ex-pressed as genome-equivalents/mL.

The results are summarized in Fig.1. We found no statistically signifi-cant differences (P 0.05, Friedmantest) among the median plasma DNAconcentrations in samples collectedinto different anticoagulants at 0, 2,and 6 h after venesection. After 24 hof whole blood incubation at roomtemperature, median plasma DNA

Table 1. Panel of paraprotein-containing specimens tested for HDL-C by theSynchron LX (Beckman) and Vitros 950 (Johnson & Johnson) assays.a

SpecimenbParaprotein

isotypeTotal

protein, g/L

Paraprotein HDL-C, mg/L

Concentration,g/L

% of totalprotein Synchron LX Vitros 950

1a IgG 122 73 60 50 1001b IgG 67 10 15 680 Not done2 IgG 116 67 58 180 2603 IgG 103 46 45 450 5504 IgG 92 43 47 420 4405 IgG 89 34 38 530 4906 IgG 77 35 45 390 3707 IgG 75 22 29 220 2008 IgG 74 16 22 320 3109 IgG 74 7 9 830 780

10 IgG 74 2 3 670 69011 IgG 102 56 55 50 27012 IgG 75 7 9 870 79013 IgG unknown 87 30 34 430 43014 IgG unknown 68 13 19 300 34015 IgA 92 24 26 490 55016 IgA 88 34 39 300 35017 IgM 101 27 27 50 26018 IgM 62 5 8 200 16019 IgM 86 21 24 380 420

a The interassay within-laboratory CV for the HDL-C assays were as follows: for the Synchron LX, 4.3% at240 mg/L and 3.3% at 660 mg/L; for the Vitros 950, 3.5% at 320 mg/L and 3.1% at 830 mg/L.

b Specimens 1a and 1b are from the same patient; specimen 1b was obtained after successful responseto multiple myeloma therapy.

256 Letters

concentrations were significantly in-creased in EDTA (940 vs 590 ge-nome-equivalents/mL; 1.6-fold; P 0.013, Wilcoxon test), heparin (4100vs 540 genome-equivalents/mL; 7.6-fold; P 0.005, Wilcoxon test), andcitrate (4300 vs 540 genome-equiva-lents/mL; 8-fold; P 0.005, Wil-coxon test) compared with their re-spective concentrations at time 0 h.We also found a statistically signifi-cant difference among the medianplasma DNA concentrations for thethree different anticoagulant types at24 h (P 0.0005, Friedman test). Inaddition, the differences in medianplasma DNA concentrations be-tween EDTA/heparin (P 0.005,Wilcoxon test) and EDTA/citrate(P 0.005, Wilcoxon test) wereboth highly statistically significant,whereas the difference in plasmaDNA concentrations between hepa-rin and citrate was not statisticallysignificant (P 0.96, Wilcoxon test).

The results from our study showthat EDTA, heparin, and citrate pro-duced similar quantitative plasmaDNA results within 6 h of venesec-tion. At 24 h after sampling, how-ever, DNA concentrations werehigher with all three anticoagulantsthan at 0 or 6 h. Therefore, highfalse-positive results are likely to oc-cur if samples are stored for 24 hbefore being analyzed. Plasma DNAoriginates at least partly from hema-topoietic cells (1 ) but does not comefrom red blood cells and plateletsbecause they are predominantlyanucleate cell populations. Increasesin plasma DNA concentrations afterstorage for 24 h are likely a result of

leukocyte necrosis or apoptosis. Theunderlying mechanism leading tothe much higher plasma DNA con-centrations from heparin and citratetubes are not clearly known. Furtherinvestigations may be needed to ex-plore on this area.

The efficiencies of the real-timePCR assays in the present study weresimilar irrespective of the choice ofanticoagulant. This is evident fromthe lack of difference in the plasmaDNA concentrations among samplescollected into tubes containing thethree anticoagulants at time 0 h. Thisfinding is different from previousobservations, where heparin was re-ported to have an inhibitory effect onPCR (46).

It is therefore acceptable to useEDTA, heparin, or citrate as the an-ticoagulant for quantitative plasmaDNA analysis provided that plasmais collected within 6 h after venesec-tion. However, EDTA is the antico-agulant of choice if delayed bloodprocessing is anticipated.

References1. Lui YYN, Chik KW, Chiu RWK, Ho CY, Lam CWK,

Lo YMD. Predominant hematopoietic origin ofcell-free DNA in plasma and serum after sex-mismatched bone marrow transplantation. ClinChem 2002;48:4217.

2. Lee TH, Montalvo L, Chrebtow V, Busch MP.Quantitation of genomic DNA in plasma andserum samples: higher concentrations ofgenomic DNA found in serum than in plasma.Transfusion 2001;41:27682.

3. Jung M, LKlotzek S, Lewandowski M, Fleis-chhacker M, Jung K. Changes in concentration ofDNA in serum and plasma during storage ofblood samples. Clin Chem 2003;49:10289.

4. Beutler E, Gelbart T, Kuhl W. Interference ofheparin with the polymerase chain reaction. Bio-techniques 1990;9:166.

5. Jung R, Lubcke C, Wagener C, Neumaier M.Reversal of RT-PCR inhibition observed in hepa-

rinized clinical specimens. Biotechniques 1997;23:248.

6. Farnert A, Arez AP, Correia AT, Bjorkman A,Snounou G, do Rosario V. Sampling and storageof blood and the detection of malaria parasitesby polymerase chain reaction. Trans R Soc TropMed Hyg 1999;93:503.

7. Lo YMD, Tein MSC, Lau TK, Haines CJ, LeungTN, Poon PMK, et al. Quantitative analysis offetal DNA in maternal plasma and serum: impli-cations for noninvasive prenatal diagnosis. Am JHum Genet 1998;62:76875.

Nicole Y.L. Lam1

Timothy H. Rainer1

Rossa W.K. Chiu2

Y.M. Dennis Lo2*

1 A&E Medicine Academic Unitand

2 Department of Chemical PathologyThe Chinese University of Hong Kong

Prince of Wales HospitalShatin, New Territories,

Hong Kong SAR

* Author for correspondence. Fax 852-2194-6171; e-mail loym@cuhk.edu.hk.

DOI: 10.1373/clinchem.2003.026013

Cross-Reactivity of ThreeRecombinant Insulin Analogs withFive Commercial InsulinImmunoassays

To the Editor:Several new insulin analogs that areprepared with recombinant DNAtechnology are available for clinicaluse [for a recent review, see Ref. (1 )].These agents have altered pharma-cokinetics compared with regularhuman insulin. Insulin aspart (Novo-LogTM; Novo Nordisk Pharmaceuti-cals) is homologous with regular hu-man insulin except for a singlesubstitution of aspartic acid for pro-line at position B28. This single sub-stitution reduces the molecules ten-dency to form hexamers. Therefore,insulin aspart is absorbed more rap-idly after subcutaneous injection andhas both a faster onset of action and ashorter duration of action than regu-lar insulin. A second short-acting re-combinant insulin is insulin lispro(Humalog; Eli Lilly and Company),which is a human insulin analog

Fig. 1. Box plots of -globinDNA concentrations in theplasma of healthy individualscollected into tubes contain-ing different anticoagulants.F, EDTA; , heparin; E, citrate.Plasma -globin DNA concentra-tions (genome-equivalents/mL)are plotted on the y axis, and thehours after venesection are indi-cated on the x axis. The upperand lower limits of the boxes andthe lines across the boxes indi-cate the 75th/25th percentilesand the medians, respectively.The upper and lower error barsindicate the 90th and 10th per-centiles, respectively. The sym-bols indicate outliers.

Clinical Chemistry 50, No. 1, 2004 257