evidence for endotoxin contamination in plastic - clinical chemistry

Evidence for Endotoxin Contamination in PlasticNa�-Heparin Blood Collection Tube Lots

Kathryn J. Newhall,1* Geoffrey S. Diemer,1 Natalia Leshinsky,1 Keith Kerkof,1 Hilary T. Chute,2

Chris B. Russell,1 William Rees,1 Andrew A. Welcher,2 Scott D. Patterson,2 and Gary D. Means1

BACKGROUND: Biomarker assays are often conducted onwhole blood samples in the course of drug develop-ment studies. Because bacterial lipopolysaccharide(LPS) (endotoxin) contamination is known to causespontaneous cytokine production by monocytes, con-tamination of blood collection tubes may interferewith biomarker assay results.

METHODS: Whole blood from healthy donors was col-lected into plastic or glass sodium (Na�)-heparinVacutainer™ blood collection tubes and heparinizedsyringes. Samples were analyzed for phosphoproteinresponse, cytokine production, and RNA expression.Tubes were tested for endotoxin contamination by useof the limulus amoebocyte lysate assay.

RESULTS: Results of phospho-flow cytometry, branchedDNA (bDNA), and ELISA assays indicated that a spe-cific lot (#5339582) of plastic Na�-heparin Vacutainertubes was highly contaminated with an endotoxinlikesubstance, and contamination was confirmed by thelimulus amoebocyte lysate assay. Analysis of multiple-analyte panels revealed that analytes whose changedexpression was predictive of LPS stimulation were in-creased when whole blood was incubated in contami-nated tubes for 6 or 18 h. Two additional lots of plastictubes tested had detectable amounts of endotoxin suf-ficient to strongly alter phospho-flow cytometry anal-yses, as determined by the fold change in phosphory-lation of p38 mitogen-activated protein kinase inresponse to tumor necrosis factor � and LPS. In con-trast, 3 lots of glass tubes had substantially lower levelsof spontaneous blood activation.

CONCLUSIONS: Endotoxin contamination associatedwith tubes from 3 lots of a particular type of plasticNa�-heparinVacutainertubedramaticallyaffectedbio-marker assay measurements. Prescreening these tubesis suggested before their use in clinical sample analysis.© 2010 American Association for Clinical Chemistry

Biomarker identification is an essential component ofdrug development. Biomarker analysis may have animpact on understanding therapeutic action of a drug,dose range (and schedule) determination, and patientresponse stratification, and allows for the targeting ofpatient populations most likely to be treated with a newdrug. The importance of biomarkers in drug develop-ment is outlined by the US Food and Drug Adminis-tration in its Critical Path Initiative (1 ). One importantgoal of the initiative is to encourage the use of measure-ments of biomarkers from patient samples to providedata on biological activities expected to correlate withclinical efficacy and safety.

Selection of appropriate specimens for biomarkermeasurement depends on the ease of sample acquisi-tion and the willingness of individuals to supply thenecessary samples. Most study participants will readilydonate blood, a situation that is advantageous becausea wide array of biomarker assays can be performed onblood samples. Assays designed to detect changes inexpression of RNA, proteins (both secreted and intra-cellular), and phosphoprotein targets are now com-monly used to analyze blood samples.

Recent technical advances have led to the applica-tion of flow cytometry for identification of intracellularphosphoprotein biomarkers. These phospho-flow cy-tometry assays have the advantage of allowing rapidassessment of changes in the phosphorylation oftarget-proximal biomarkers, typically within minutesof pathway activation. In these assays, the effect of per-turbation may be measured by using fluorescently la-beled monoclonal antibodies to identify changes inspecific phosphoprotein concentrations. These assaysare read in a cell-intrinsic fashion and therefore are asrobust for rare cell types as for abundant cellpopulations.

For any biomarker assay, it is critical that the bloodcollection procedure not affect the readout. Substantialeffort is expended to test and qualify assays, yet equalattention must be applied to preanalytical variables.Analysis of errors in laboratory medicine has demon-

1 Amgen Molecular Sciences, Amgen, Seattle, WA and 2 Thousand Oaks, CA.* Address correspondence to this author at: 1201 Amgen Court West, Seattle, WA

98119. Fax 206-217-0346; e-mail [email protected].

Received February 2, 2010; accepted June 15, 2010.Previously published online at DOI: 10.1373/clinchem.2006.144618

Clinical Chemistry 56:91483–1491 (2010)

General Clinical Chemistry

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strated that a high percentage of errors (30%–75%)occur in the preanalytical phase rather than in the an-alytical phase (2–5 ). Preanalytical variables to considerinclude sample collection, handling, processing, stor-age, and transport to the analytical laboratory. It istherefore critical that standardized protocols be devel-oped and submitted to the clinical sites where biomar-ker samples are collected. These protocols must be verydetailed to prevent preanalytical errors. In fact, one ofthe more commonly reported sources of erroneousmeasurements has been the use of the wrong type ofcollection tube (3 ). In this study we examined the mag-nitude of measurement errors that might also easilyarise as a result of variable blood activation within asingle type of collection tube.

We tested and qualified assays with respect to arange of preanalytical variables, such as the effects ofanticoagulants on whole blood assays. Because the che-lation of calcium (Ca��) ions, which occurs with anti-coagulants such as citrate and EDTA, affects someCa��-sensitive signal transduction pathways, wechose to develop the majority of our assays in wholeblood collected in Na�-heparin. Here we report ourfindings that endotoxinlike contamination associatedwith tubes from 3 lots of a particular type of plasticNa�-heparin Vacutainer blood collection tubes dra-matically affected the outcome of biomarker assaymeasurements by flow cytometry, ELISA, and RNAanalysis.

Materials and Methods

WHOLE BLOOD COLLECTION

Whole blood from healthy donors was collectedthrough the Amgen Blood Donor Program (Seattle,WA, and Thousand Oaks, CA). This program is con-ducted in accordance with the Declaration of Helsinkiand the International Conference on HarmonizationGood Clinical Practice Guidelines and meets compli-ance requirements established by the Western Insitu-tional Review Board. All donors gave written informedconsent.

WHOLE BLOOD STIMULATIONS AND FIXATION

Blood was collected in 10-mL Na�-heparin plastic[Becton Dikinson (BD),3 cat #367874, lots #5339582,#6123786] or glass (BD, cat #366480, lot #5007997)

Vacutainer tubes or 60-mL syringes (BD, cat #309653)containing certified endotoxin-free Na�-heparin(10 000 U/mL, 1 �L/mL whole blood; Sigma-Aldrich,cat #H3393). Tumor necrosis factor � (TNF-�)(R&D Systems, cat #210-TA/CF, lot #AA335111, andAA335054) and lipopolysaccharide (LPS) (Sigma-Aldrich, cat #L6529, lot #015K4103, and lot#016K4021) were diluted in PBS � 0.1% endotoxin-free BSA (Calbiochem, EMD Chemicals, Merck KGaA,cat # 126579). From each of the collection tubes 250 �Lof whole blood was stimulated with TNF-� (2.5 �g) for5 min or LPS (25 �g) for 7.5 min at 37 °C. After thetreatment times elapsed, 5 mL of prewarmed (37 °C)1� BD Phosflow Lyse/Fix Buffer (BD Biosciences, cat#558049) was added to each sample to simultaneouslylyse the red blood cells (RBCs) and fix the peripheralblood mononuclear cells (PBMCs). The Lyse/FixBuffer–treated sample tubes were incubated for 20 min ina 37 °C water bath to allow for complete RBC lysis. Afterincubation the tubes were centrifuged at 1000g for 15 minat 4 °C to pellet the fixed whole blood PBMCs.

FLOW CYTOMETRY

The pelleted PBMCs were washed 3 times with ice-coldPBS � 1% FBS, with pelleting by centrifugation per-formed each time. Extracellular antigen detection wasconducted for 30 min by using a cocktail of CD14-Pacific Blue (BD Biosciences, cat #558121) and NK Si-multest™: CD3-FITC, CD56/16-PE (BD Biosciences,cat #340042) diluted in PBS � 1% FBS. After beinglabeled, the cells were pelleted and washed 3 times inPBS � 1% FBS. Cells were then permeablized with 200�L ice-cold 80% methanol per sample and incubatedon ice for 20 min in the dark. Cells were againwashed 3 times before intracellular antigen detec-tion. Cells were then labeled with a mouse antihu-man mitogen-activated protein kinase (MAPK)phospho-p38-AlexaFluor 647 antibody (BD Bio-sciences, cat #612595) diluted in PBS � 1% FBS. Cellswere washed 3 times and resuspended in PBS � 1%FBS for analysis, performed by using the LSRII BDFlow Cytometer system with 4-color analysis. CD14-positive cells were gated and specifically analyzed forphospho-p38-AF647 fluorescence.

CYTOKINE PRODUCTION ASSAY AND MEASUREMENT OF

INTERLEUKIN-1� AND TNF-�

Whole blood from 2 healthy donors was collected in10-mL Na�-heparin plastic (BD, cat #367874, lot#5339582) Vacutainer tubes, 2 different Na�-heparinglass Vacutainer tubes (BD, cat #366480, lot#5311775), or Monoject tubes (Covidien, cat #320751,lot #312120). LPS (List Biological Laboratories, cat #201) was diluted in assay medium. Assay medium con-sisted of RPMI 1640 (Invitrogen Life Technologies

3 Nonstandard abbreviations: BD, Becton Dickinson; TNF-�, tumor necrosis factor�; LPS, lipopolysaccharide; RBC, red blood cells; PBMC, peripheral bloodmononuclear cells; MAPK, mitogen-activated protein kinase; IL, interleukin;MAP, multiple analyte panel; CCL, chemokine ligand; MCP, monocyte chemo-tactic protein; PTX3, pentraxin 3; bDNA, branched DNA; LAL, limulus amoebo-cyte lysate.

1484 Clinical Chemistry 56:9 (2010)

Corporation, cat #11875) containing 1� penicillin/streptomycin/glutamine (Invitrogen, cat #10378) and0.1% human serum albumin (Bayer AG, cat #692–20).Cultures of 50% whole blood/50% assay medium in afinal volume of 200 �L were incubated for 18 h at 37 °Cand 5% CO2 in the presence of 0 or 100 �g/L LPS in a96-well tissue-culture plate (BD Biosciences, cat#353072). LPS stimulation was initiated �1 h afterblood draw and samples were run as triplicates. Afterincubation, the assay plates were centrifuged at 4 °C for15 min at 1500g, and 100 �L of supernatant was trans-ferred to a polypropylene plate for storage at �80 °Cuntil analysis for interleukin (IL)-1� and TNF-� couldbe performed. IL-1� and TNF-� were assayed by usingthe custom-coated MSD 96-well Multi-Array® HumanCytokine Assay Ultrasensitive kit (Meso Scale Discov-ery, cat #N41IB-1) according to the manufacturer’s kitinstructions for serum/plasma samples. Data were an-alyzed in GraphPad Prism 4.0 (GraphPad Software) byusing sigmoidal dose response (variable slope) with1/y2 weighting. The CV% was determined by (SD/mean) � 100.

Whole blood from 3 separate donors was also col-lected in either glass (BD, cat #366480, lot #5311755)or plastic Na�-heparin Vacutainer tubes (BD, cat#367874, lot #5339582) and allowed to incubate for 6or 18 h. Plasma was removed and analyzed by using amultiple analyte panel (MAP) (Rules Based Medicine).

BRANCHED-DNA ASSAY

Cultures of 80% whole blood in RPMI medium werestimulated for 24 h in 96-well microtiter plates at 37 °Cand 5% CO2 in the presence of 3 �g/L TNF-� (R&DSystems, cat #210-TA) and 0 –1000 �g/L IL-17 (R&DSystems, cat #317-ILB). Heparinized whole blood fromAmgen Washington Blood Donor Program volunteerswas collected into either plastic (lot #5339582) or glass(lot #5007997) BD Na�-heparin Vacutainer tubes, andthe cytokine stimulation was initiated �3 h after bloodharvest.

Blood cell lysis and quantification of chemokineligand (CCL) 2/monocyte chemotactic protein (MCP)1, CCL7, pentraxin 3 (PTX3), and IL-6 mRNA wereperformed as outlined by the manufacturer of thebranched DNA (bDNA) assay kit (Panomics). Briefly,20 �L of each blood culture was transferred to a deep-well, 96-well plate and lysed after the addition of 76 �Lof a proprietary lysis solution from Panomics to eachwell and incubation at 60 °C for 1 h with shaking at 100rpm. Lysate from each well was hybridized with DNAprobes specific for the genes for CCL2, CCL7, PTX3,and IL-6 and capture beads for 16 h at 58 °C with shak-ing at 600 rpm. Capture beads hybridized to DNAprobes and lysate RNA were washed on a vacuum-assisted 96-well filter plate apparatus and further hy-

bridized for 1 h at 54 °C with shaking at 600 rpm first toan Amplifier Diluent and subsequently to a biotinyl-ated DNA Label Probe provided in the kit, withwashing steps before and after the Label Probe hy-bridization. The capture beads ultimately associatedwith biotinylated label probe were labeled withphycoerythrin-conjugated streptavidin at room tem-perature for 30 min with 600 rpm shaking. After 2 ad-ditional washing steps, the fluorescence intensity of thelabeled beads in each well of the 96-well plate was eval-uated on a LiquiChip 200 Workstation (Qiagen).

LIMULUS AMOEBOCYTE LYSATE ASSAY

For the limulus amoebocyte lysate (LAL) assay,endotoxin-free magnesium chloride (MgCl2) bufferwas added to 6 different lots of Vacutainer tubes: plas-tic Vacutainer tubes (BD cat #367874, lots #5339582,6123786, and 6100968) and glass Vacutainer tubes (BDcat #367874, lots #5007997, 5311755, and 6123675).Buffer was allowed to incubate in the tube for 24 hbefore endotoxin analysis using the Kinetic-QCL kit(Lonza).

Results

One assay for monitoring the activation of TNF recep-tor 1 in whole blood is the measurement of TNF-�–induced increased phosphorylation of p38 MAPK (Fig.1). During routine biomarker development assays, thispreviously qualified assay began to perform erraticallyor failed completely. An extensive evaluation of re-agents and buffers was conducted but yielded no in-sight into the apparent loss of activity. At this point, werecognized that the sample collection tubes for ourwhole-blood– based assays had been switched fromglass (lot #5007997) to plastic Na�-heparin Vacutainertubes (lot #5339582).

To compare the 2 types of blood collection tubes,we used a phospho-flow cytometry protocol to assaywhole blood monocytes (which are very sensitive toendotoxin) for an increase in activated p38 MAPK inresponse to either TNF-� or LPS. Whole blood wascollected in heparinized syringes, 10 mL Na�-heparinglass tubes, or the 10 mL Na�-heparin plastic tubesand stimulated with PBS alone, PBS containing 0.1%BSA, 2 different lots of LPS (100 �g/L), or 2 differentlots of TNF-� (10 �g/L).

Although baseline phosphorylation of p38 MAPKwas similar in monocytes from blood collected in eitherglass or plastic Vacutainer tubes (Fig. 1), TNF-� treat-ment failed to induce phosphorylation of p38 MAPK inmonocytes from whole blood collected in plastic tubes.In contrast, we observed a 3.5-fold increase in phos-phorylated p38 in TNF-�–stimulated blood from theglass tubes or heparinized syringes. LPS stimulation

Endotoxin Contamination in Vacutainer Tubes

Clinical Chemistry 56:9 (2010) 1485

did lead to a 2.5-fold increase in p38 phosphorylationin stimulated blood collected in plastic tubes. However,this response was blunted compared with the LPS-induced stimulation of phosphorylated p38 in bloodfrom the glass tubes or heparinized syringes (5.4-foldincrease). Different lots of TNF-� and LPS had similareffects on p38 phosphorylation regardless of the bloodcollection method used, indicating that these reagentswere not contaminated.

In a concurrent set of bDNA-based assays, unex-pected increases in IL-6, CCL7, PTX3, and CCL2mRNA expression were observed in whole blood notstimulated with exogenous cytokines. In many re-spects, the results obtained were similar to that ob-served with LPS stimulation. To test the effects of bloodcollection tubes on the bDNA assay, whole blood froma single donor was drawn into either glass (lot#5007997) or plastic (lot #5339582) Vacutainer tubes.An increase in the basal expression of CCL2, CCL7,IL-6, and PTX3 mRNA was observed when blood wascollected into plastic tubes (Fig. 2). LPS increasedmRNA expression of these cytokines in blood collectedinto glass tubes but no additional increase was ob-served in blood collected in plastic tubes.

We assessed the production of 2 cytokines (IL-1�and TNF-�) known to be induced in whole blood ex-posed to LPS to confirm that the observed effects onp38 phosphorylation in whole blood collected in plas-tic Vacutainer tubes was affecting events downstreamof signal transduction. Blood was collected from 2 dif-ferent donors into plastic Na�-heparin Vacutainertubes (lot #5339582), glass Na�-heparin Vacutainertubes (lot #5311755), or plastic Monoject® blood col-lection tubes (Kendall, lot #312120). Cytokine produc-tion was measured by ELISA (Table 1). IL-1� produc-tion from blood collected in the glass Vacutainer tubesor the Monoject tubes was below the lower limit ofquantification of the assay (1.56 ng/L; Table 1). In con-trast, blood from both donors collected in the plasticVacutainer tubes displayed increased IL-1� concentra-tions, ranging from 2.4 �g/L to above the upper limit ofquantification of the assay. Similarly, TNF-� produc-tion was below the lower limit of quantification inblood from both donors collected in the glass Vacu-tainer tubes or the Monoject tubes, but TNF-� concen-trations were increased in blood collected by using theplastic Vacutainer tubes. TNF-� production in bloodfrom the plastic tubes ranged from 2 to 3 �g/L (Table 1).

To further investigate the analytes affected by thecollection tube choice, we analyzed blood using theMAP. Whole blood from 3 separate donors was col-lected into glass or plastic Na�-heparin Vacutainertubes (lot #5339582) and allowed to incubate for 6 or18 h. Plasma was removed and analyzed using theMAP. The expression of at least 20 different analyteswas increased when blood was collected in the plastictubes (Fig. 3). Most of the analytes that were increasedin the blood collected in the plastic tubes were alsoincreased when the blood was stimulated with LPS ateither 6 or 18 h (data not shown). Analytes such asTNF-�, MCP-1/CCL2, and IL-6, which would be pre-dictive of LPS stimulation, were increased. However,there were also some analytes that had an unexpectedincrease as a result of LPS stimulation or suspected en-

Fig. 1. Plastic Vacutainer tubes tolerize againstTNF-�–induced phosphorylation of MAPK p38.

Two lots of TNF-� and LPS were tested against controlsin whole blood collected from a single donor into aplastic Vacutainer (lot #5339582), a heparinized syringe,and a glass Vacutainer (lot #5007997). Histograms de-pict LPS- and TNF-�–induced phosphorylation of MAPKp38 (P-p38) in CD14� monocytes. MFI, median fluores-cence intensity.


dotoxin contamination. These included �-fetoproteinand brain-derived neurotrophic factor. These resultsfurther support the likelihood that the contaminant inthe plastic tubes was endotoxin.

Taken together, the results of the phospho-flowcytometry data, the bDNA data, and the cytokine pro-duction data all indicate that the particular lot of10-mL plastic Na�-heparin tubes we used was contam-inated with endotoxin or a substance that acted in amanner similar to endotoxin. At this point we tested

additional lots of Vacutainer tubes for evidence of sim-ilar activity. In an attempt to quantify the concentra-tions of endotoxins in the different tube lots, we addedendotoxin-free MgCl2 buffer to 6 different lots of Va-cutainer tubes. Three lots of plastic Vacutainer tubes(BD cat #367874, lots #5339582, 6123786, and6100968) and 3 lots of glass Vacutainer tubes (BD cat#367874, lots #5007997, 5311755, and 6123675) weretested for endotoxin by using the LAL assay. As indi-cated in Table 2, endotoxin concentrations varied

Fig. 2. Baseline mRNA levels are increased in bDNA-based assays conducted on whole blood collected in plasticVacutainer tubes.

Whole blood from an individual donor was collected in plastic (lot #5339582) or glass Vacutainer tubes (lot #5007997). Thebasal expressions of CCL2, CCL7, IL6 and PTX3 mRNA were increased when collected in plastic tubes. LPS (1 �g/L) additionto whole blood collected in glass tubes resulted in increased expression of all 4 mRNAs, whereas no further increase was notedin whole blood collected in plastic tubes.

Table 1. IL-1� and TNF-� production as a function of blood collection tubes.a

Donor Vendor Cat no. Lot

IL-1� TNF-�

Concentration, ng/L CV% Concentration, ng/L CV%

1488 BD 366480 5311755 �15.6 — �15.6 —

1488 Monoject 320751 312120 �15.6 — �15.6 —

1488 BD 367874 5339582 2842 10.1 2309 5.0

1488 BD 367874 5339582 2351 19.5 2239 18.7

1492 BD 366480 5311755 �15.6 — �15.6 —

1492 Monoject 320751 312120 �15.6 — �15.6 —

1492 BD 367874 5339582 8174 4.1 2592 12.5

1492 BD 367874 5339582 Above ULOQ — 3386 14.8

a Whole blood from 2 donors was collected into BD plastic and glass Na�-heparin Vacutainer tubes and plastic Monoject collection tubes and allowed to incubatefor 6 h. IL-1� and TNF-� were assayed by using ELISA. ULOQ, upper limit of quantification.



widely in plastic tubes tested in this assay, ranging from0.53 to 7.7 k endotoxin units/L. Although the concentra-tions varied, all 3 lots of plastic tubes that were tested haddetectable concentrations of endotoxin. It is not known ifthe variation was due to the LAL assay or the tubes them-selves. In contrast, endotoxin concentrations were belowthe limit of detection in all the glass tubes tested exceptone, and it is possible that this increase in endotoxin wasdue to contamination during the LAL assay.

In a final effort to compare the sensitivities of themethods used to test for possible endotoxin contami-nation, we identified a plastic Vacutainer tube lot thatseemed to be uncompromised according to results ofIL-1� and TNF-� expression measured after incuba-tion (see Table in the Data Supplement that accompa-nies the online version of this article at http://www.clinchem.org/content/vol56/issue9). This lot of plasticVacutainer tubes induced the lowest cytokine increase

Fig. 3. Analyte expression in unstimulated whole blood.

Whole blood was collected from 3 donors in either glass or plastic tubes and incubated for 6 or 18 h. Plasma was removed andanalyzed by using the MAP. Fold changes vs the 6-h control are shown within each heat-map panel. Yellow, increase frombaseline; blue, decrease from baseline; white, no change from baseline. MIP, macrophage inflammatory peptide; ENA, epithelialneutrophil-activating protein; G-CSF, granulocyte colony-stimulating factor.


we had observed in any of the lots of plastic tubes(�1% of that observed in the severely contaminatedlot). The small amount of contamination present thatinduced slight changes in IL-1� and TNF-� was con-firmed by the effects on both basal and induced p38phosphorylation (Fig. 4). In this case the phospho-flowcytometry analysis was sufficiently sensitive to bestrongly affected by even a very low amount of contam-inating endotoxin as measured by the fold change inthe phosphorylation of p38 in response to eitherTNF-� or LPS. Therefore, even low amounts of con-

tamination can have a profound effect, depending onthe technologies used and on the analytes measured.

Discussion

It has been recognized since the 1980s that bacterialLPS (endotoxin) contamination results in the sponta-neous production of cytokines by monocytes (6, 7 ).Efforts were subsequently made to produce endotoxin-free reagents for use in cellular immunological assays.However, these reagents are useless if the sample col-

Table 2. Summary of endotoxin concentrations in plastic and glass Vacutainer tubes.a

Tubesample

Lotnumber

Tubetype

LAL,kEU/L Tube sample

Lotnumber

Tubetype

LAL,kEU/L

1 5339582 Plastic 2.13 1 5007997 Glass �0.50

2 5339582 Plastic 1.38 2 5007997 Glass �0.50

3 5339582 Plastic 1.95 3 5007997 Glass �0.50

4 5339582 Plastic 7.71 4 5007997 Glass �0.50

5 5339582 Plastic 6.85 5 5007997 Glass �0.50

6 5339582 Plastic 6.38 6 5007997 Glass �0.50

7 5339582 Plastic 6.41 7 5007997 Glass �0.50

8 5339582 Plastic 7.42 8 5007997 Glass �0.50

9 5339582 Plastic 0.98 9 5007997 Glass �0.50

10 5339582 Plastic 0.71

11 5339582 Plastic �0.50 1 5311755 Glass �0.50

12 5339582 Plastic 0.66 2 5311755 Glass �0.50

13 5339582 Plastic 0.98 3 5311755 Glass �0.50

4 5311755 Glass �0.50

1 6100968 Plastic 0.65

2 6100968 Plastic �0.50 1 6123675 Glass �0.50

3 6100968 Plastic 1.15 2 6123675 Glass �0.50

4 6100968 Plastic 0.55 3 6123675 Glass �0.50

5 6100968 Plastic 0.74 4 6123675 Glass �0.50

6 6100968 Plastic �0.50 5 6123675 Glass �0.50

7 6100968 Plastic �0.50 6 6123675 Glass �0.50

7 6123675 Glass 2.876

1 6123786 Plastic �0.50 8 6123675 Glass �0.50

2 6123786 Plastic 0.54

3 6123786 Plastic �0.50

4 6123786 Plastic 0.62

5 6123786 Plastic 0.57

6 6123786 Plastic �0.50

7 6123786 Plastic �0.50

8 6123786 Plastic �0.50

a Endotoxin-free MgCl2 buffer was added to 6 different lots of Vacutainer tubes. Three lots of plastic Vacutainer tubes (BD cat #367874, lot #5339582, #6123786,and #6100968) and 3 lots of glass Vacutainer tubes (BD cat #367874, lot #5007997, #5311755, and #6123675) were tested for endotoxin by using the LAL assay.EU, endotoxin units.



lection tubes are contaminated. Sporadic reports ofcontamination in blood collection tubes can be foundin the literature. It was previously reported that 10-mLNa�-heparin Vacutainer tubes contained a mean (SD)LPS/tube of approximately 315 (95) pg as measured bythe LAL assay (8 ). In addition, tubes contaminatedwith endotoxin can stimulate cytokine production inwhole blood. Vacutainer tubes containing contami-nated lithium heparin strongly stimulated IL-1, IL-6, andTNF-� production in whole blood after as little as 2 h ofincubation (9). EDTA Vacutainer tubes or tubes contain-ing endotoxin-free heparin had no effect on cytokine pro-duction, indicating that the choice of anticoagulant wasnot the factor contributing to spontaneous cytokineproduction. Another group found similar results whenwhole blood was collected in Na�-heparin Vacutainertubes that contained 10 ng/L of endotoxin (10 ).

The results of this study also mirror the well-documented phenomenon of endotoxin tolerance. Inendotoxin tolerance, repeat exposure to endotoxin al-ters the signaling network within a cell, leading to amarked decrease in responsiveness to LPS stimulation

(11 ). This hyporesponsiveness is usually manifested asdiminished secretion of cytokines, particularly TNF-�and IL-6 (12 ). However, changes in signaling mole-cules downstream of toll-like receptor 4 have also beenobserved (11 ). In the current study, we observedchanges in both signaling, as detected by phospho-flowcytometry, and cytokine release in whole blood as aresult of prolonged exposure to endotoxin in the con-taminated tubes. This in vitro tolerance alters the in-flammatory response in the cells and therefore masksthe overall patient response, making it impossible todraw conclusions from clinical trials.

Our data point to the conclusion that there may bea systemic problem with the use of plastic Na�-heparinVacutainer tubes, in that blood collected in any of 3 lotsof extensively tested tubes consistently behaved as ifstimulated with endotoxin. Endotoxins stimulate cel-lular responses through the toll-like receptor system inblood, which is sensitive to a wide variety of chemicalsthat often resemble molecules released from patho-gens. The observed effects could therefore be due tocontamination with endotoxin during the manufac-

Fig. 4. The phospho-flow assay is sufficiently sensitive to detect very low amounts of contaminating endotoxin.

Basal phosphorylation was higher and LPS or TNF-� stimulation was less robust in monocytes from whole blood collected inplastic tubes without detectable endotoxin (lot #6123786) (as determined by cytokine measurement) compared to bloodcollected in glass tubes (lot #5007997). MFI, median fluorescence intensity; P-p38, MAPK p38 phosphorylation.


turing process, or possibly even to cellular recognitionof other synthetic chemicals incorporated into the plas-tic and then released into the blood. Highly sensitivephospho-flow cytometry assays are useful for evaluat-ing drug development projects and determining on-target assessments, which inform the further clinicaldevelopment of drug candidates. These kinds of assaysare also critical for understanding disease etiology on amolecular level. They provide on-target assessment oftherapeutic processes, and have even been used to pro-vide information used to determine patient-dosingconcentrations. The observation that the gold standardLAL assay and the relatively more sensitive cytokinedetection assays were unable to detect low-level endo-toxin contamination illustrates the need for rigorousevaluations of novel approaches for identifying andevaluating new whole blood biomarkers becausestrong responses are readily detectable with the use ofthe more sensitive phospho-flow cytometry assay. Aswe continue to develop these powerful and innovativeassays it is necessary to keep a critical eye on sources ofvariation that may be introduced by something as os-tensibly innocuous as the type of blood collection tubeused. Caution is recommended in assessing the poten-tial effects of this preanalytical variable before collect-ing and analyzing clinical samples.

Author Contributions: All authors confirmed they have contributed tothe intellectual content of this paper and have met the following 3 re-quirements: (a) significant contributions to the conception and design,acquisition of data, or analysis and interpretation of data; (b) draftingor revising the article for intellectual content; and (c) final approval ofthe published article.

Authors’ Disclosures of Potential Conflicts of Interest: Uponmanuscript submission, all authors completed the Disclosures of Poten-tial Conflict of Interest form. Potential conflicts of interest:

Employment or Leadership: K.J. Newhall, Amgen; N. Leshinsky,Amgen; C.B. Russell, Amgen; W. Rees, Amgen; A.A. Welcher, Am-gen; S.D. Patterson, Amgen; and G.D. Means, Amgen.Consultant or Advisory Role: None declared.Stock Ownership: K.J. Newhall, Amgen; N. Leshinsky, Amgen; H.T.Chute, Amgen; C.B. Russell, Amgen and Caliper Life Sciences;W. Rees, Amgen; A.A. Welcher, Amgen; S.D. Patterson, Amgen; andG.D. Means, Amgen.Honoraria: None declared.Research Funding: N. Leshinsky, Amgen; G.D. Means, Amgen.Expert Testimony: None declared.

Role of Sponsor: The funding organizations played no role in thedesign of study, choice of enrolled patients, review and interpretationof data, or preparation or approval of manuscript.

Acknowledgments: The authors gratefully acknowledge the assis-tance of A. Newman and J. Walker in performing the LAL assays.

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evidence for endotoxin contamination in plastic - clinical chemistry

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