Thrombosis Research 126 (2010) Contents lists available at ScienceDirect. Thrombosis Research

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1 Thrombosis Research 126 (2010) Cotets lists available at ScieceDirect Thrombosis Research joural homepage: Regular Article Moitorig treatmets with ufractioated hepari: CTAD must be used istead of citrate as the aticoagulat solutio whe usig partial-draw collectio tubes. Results of a multiceter evaluatio Pierre Toulo a,, Liê Abecassis b, Motalib Smahi c, Catherie Terisie d a Uiversité Nice Sophia-Atipolis, Faculté de Médecie, CHU Sait Roch, Service d'hématologie Biologique, Nice, Frace b Service de Biologie, Cetre Hospitalier R Ballager, Aulay sous Bois, Frace c Laboratoire d'hématologie, Hôpital Simoe Veil, Eauboe-Motmorecy, Frace d Laboratoire d'hématologie, CHU Hôtel-Dieu,Nates, Frace article ifo abstract Article history: Received 8 February 2010 Received i revised form 11 August 2010 Accepted 22 August 2010 Available olie 14 October 2010 Keywords: Collectio tubes Citrate CTAD Hepari therapy Moitorig Backgroud: Samplig small volumes of blood may be ecessary, particularly i pediatric patiets, or i case of difficult or recurret veipuctures. Methods: Routie hemostasis test results evaluated i partial- ad full-draw evacuated polymer tubes obtaied i 4 ceters were compared. Results: No relevat discrepacy (Blad-Altma) was foud betwee test results measured i partial- ad full-draw tubes obtaied from utreated patiets ad from patiets o vitami K-atagoist or low molecular weight hepari. I patiets o ufractioated hepari (UFH), sigificatly lower ati-fxa activity [media=0.29 IU/mL (rage: ) vs ( ), =89, p b0.0001] ad shorter aptt were measured i partial-draw tubes. This discrepacy was likely to be related to the release of higher amouts of PF4 after icreased platelet activatio i partial-draw tubes. As CTAD is kow to couteract platelet activatio, we the collected blood ito partial-draw CTAD tube ad full-draw. Both i patiets o UFH ad i utreated patiets, o relevat differece could be demostrated for all studied parameters (Blad-Altma), icludig aptt ad ati-fxa activity, eve if aalytical compariso showed sigificatly higher ati-fxa activity i partial-draw CTAD tha i full-draw citrated tubes with a mea bias of 0.02 IU/mL, idetical throughout the measurig rage. Coclusios: These results suggest that samples collected ito partial-draw s allow accurate routie coagulatio testig i all patiets but those requirig UFH assessmet, i which their use led to a sigificat uderestimatio of aticoagulatio. I such cases, partial-draw tubes cotaiig CTAD could be validly used to moitor hepari therapy as well as to perform routie coagulatio testig Published by Elsevier Ltd. Itroductio Abbreviatios: Ati-FXa, ati-activated factor X (activity); ANOVA, aalysis of variace; aptt, activated partial thromboplasti time; CTAD, citrate, theophyllie, adeosie, dipyridamole; CV, coefficiet of variatio; ELISA, ezyme-liked immuosorbet assay; FV, (coagulatio) factor V; FVIII, (coagulatio) factor VIII; INR, iteratioal ormalized ratio; IU, iteratioal uit; LMWH, low molecular weight hepari (derivative); PET, polyethylee terephtalate; PF4, platelet factor 4; PT, prothombi time; SD, stadard deviatio; SC, subcutaeous; UFH, ufractioated hepari; VKA, vitami K atagoist. This study was preseted i part at the 51st Aual Meetig of the America Society of Hematology (New Orleas, LA, USA, December 5-8, 2009) ad appeared i abstract form i Blood 2009; 114: Correspodig author. CHU, Hôpital de Cimiez, Service d'hématologie Biologique, 4 aveue de la Reie Victoria, BP 1179, F Nice Cedex 1, Frace. Tel.: ; fax: address: toulo.p@chu-ice.fr (P. Toulo). Samplig small volumes of blood may be ecessary, particularly i pediatric patiets, or i case of difficult or recurret veipuctures. To achieve that goal, differet optios are available, depedig o the collectio tube maufacturers. Eve if the use of tubes with a smaller tha usual exteral volume is possible, the global tred toward higher stadardizatio ad laboratory automatio is i favor of usig cotaiers with the same exteral dimesio tha the usual oes but allowig to collect lower blood volumes. Beside the possibility to use a reduced iteral volume by icreasig the tube thickess, most of the maufacturers propose tubes with the same iteral dimesio but with a reduced vacuum allowig the withdrawal of a smaller blood volume. I those partial-draw tubes, the volume of citrate aticoagulat solutio has to be adapted to esure the 9 vol./1 vol. blood-to-aticoagulatio solutio volumes ratio /$ see frot matter 2010 Published by Elsevier Ltd. doi: /j.thromres

2 P. Toulo et al. / Thrombosis Research 126 (2010) If a large body of literature is dedicated to the compariso of socalled plastic tubes vs. glass tubes [1 15] ad to the impact of uderfillig citrate collectio tubes [16 20] o coagulatio test results, very few publicatios evaluated the potetial impact of collectig blood ito commercially available partial-draw collectio tubes. Actually, ad to the best of our kowledge, oly two publicatios dealig with blood collectio for coagulatio testig ito partial-draw tubes are available so far [21,22]. Both focused o the potetial impact of their use i patiets treated with ufractioated hepari (UFH) ad cocluded that ufilled tubes were usuitable for moitorig treatmets with UFH [21,22]. Oe was published by a Australia group which evaluated uusual 6.7 ml collectio tubes filled with 4.5 or 1.8 ml blood (0.11 M citrate aticoagulatio solutio volume was 0.5 ad 0.2 ml respectively) [21]. The other was about stadard 5 ml evacuated tubes of ukow origi ad material cotaiig either 0.3 ml or 0.5 ml 3.8% citrate aticoagulatio solutio volume ad a vacuum adapted to allow collectio of either 2.7 or 4.5 ml blood respectively [22]. The aim of the preset multiceter study, ivolvig four hematology laboratories, was to compare hemostasis test results obtaied i tubes from a sigle maufacturer ad specifically desiged to have a omial volume of 2.0 ml (partial-draw tubes) or a 4.0 ml (full-draw tubes), that were collected from patiets treated or ot with ay aticoagulat therapy. Patiets, Materials, ad Methods Descriptio of the tubes I the first part of the study, we evaluated two differet evacuated polymer tubes cotaiig M citrate as the aticoagulat, specifically desiged by the maufacturer (Terumo Europe, Leuve, Belgium) to have a omial volume of 2.0 ml ad 4.0 ml, as defied accordig to the ISO 6710 orm [23]. Both, made of the same material (polyethylee terephtalate, PET) ad closed with idetical rubber stopper ad safety cap, had the same exteral ad iteral dimesios. The VeoSafe TM VF-052SBCS07 cotaied 0.20 ml of a M (3.2%) sodium citrate solutio [24] ad the vacuum is adapted to allow 1.8 ml blood to be collected (partial-draw tubes). The VeoSafe TM VF-054SBCS07 cotaied 0.4 ml of a M (3.2%) sodium citrate as the aticoagulatio solutio ad a vacuum defied to allow the collectio of 3.6 ml blood (full-draw tubes). The ratio of blood to sodium citrate aticoagulat solutio was 9:1 i both partialad full-draw tubes. They were provided packed i a special sealed alumium bag cotaiig 10 tubes each, wherefore the maufacturer claims that it couteracts the evaporatio of liquid from the tube as well as the iflux of air ito the tube, allowig a shelf life of 18 moths as stated i the package isert. Sigle lots of partial- (lot # , exp. 09/07) ad full-draw (lot # , exp. 09/06) tubes were used i all the ceters throughout the first part of the study. I the secod part of the study, i additio to the VeoSafe VF- 054SBCS07 full-draw, we evaluated aother partial-draw evacuated polymer tube cotaiig a mixture of aticoagulat ad atiplatelet agets i.e mmol/l tri-na citrate, 27.5 mmol/l citric acid, 15 mmol/l theophyllie, 3.74 mmol/l adeosie, ad 0.20 mmol/l dipyridamole (CTAD) as the aticoagulat solutio, from the same maufacturer (VeoSafe VF-052SCTAD, Terumo Europe). It was idetical to the previously described VeoSafe s regardig material (PET), rubber stopper, safety cap, exteral ad iteral dimesios, ad was provided i the same packagig cotaiig 10 uits, as i the case of s. The VeoSafe VF-052SCTAD cotaied 0.20 ml of the CTAD solutio ad the vacuum is adapted to allow oly 1.8 ml blood to be collected (partial-draw tubes) leadig to a omial volume of 2.0 ml. Sigle lots of partial-draw CTAD tubes (lot # , exp. 10/03) ad full-draw s (lot # , exp. 10/02) were used i all of the ceters throughout the secod part of the study. Patiets Blood samples were obtaied i the four participatig ceters from a total of 357 adult patiets who were prescribed coagulatio testig. Eighty four patiets were treated with vitami K-atagoists (VKA). Eighty ie patiets were treated with UFH ad 48 patiets with subcutaeous (SC) ijectios of low molecular weight hepari derivatives (LMWH) for prophylaxis purpose or with full dose regime. All of the patiets o LMWH were treated with eoxapari but three who received full dose regime of tizapari (175 IU/kg SC oce a day) for the treatmet of pulmoary embolism. All patiets o UFH or LMWH had detectable amouts of ati-fxa activity i.e IU/mL. Samples from patiets without ay aticoagulat treatmet (=136) were also studied, icludig 11 hemophilia A patiets either substituted or ot with clottig factor cocetrate. I the secod part of the study, blood samples were obtaied i the four participatig ceters from a total of 204 adult patiets. Niety eight patiets were treated with UFH ad had ati-fxa activity 0.05 IU/mL, whereas 106 patiets were without ay aticoagulat treatmet, icludig 8 hemophilia A patiets either substituted or ot with clottig factor cocetrates. Usually, oe partial- ad oe full-draw tubes were collected from adult patiets requirig coagulatio testig, before beig set to the local hemostasis laboratory where they were aalyzed simultaeously for routie coagulatio tests. I additio, i a prelimiary study desiged to evaluate the time-depedat variability of test results, 4 partial- ad 4 full-draw s were collected i a radom order from 10 healthy voluteers. Oly those samples with a fill volume of more tha 90% of their omial volume were aalyzed. I all cases, patiets aged below 18 years old, kow aemic patiets (hemoglobi below 8.0 g/dl) ad pregat wome were excluded. The study was performed i accordace of the Declaratio of Helsiki, after beig approved by the local Ethics Committee. Materials ad Methods Plasma, obtaied by cetrifugatio at 3,000 x g at 12 C for 15 mi. accordig to the curret recommedatio [25,26],was aalyzed for the differet hemostasis tests withi 2 hours. However, i the prelimiary study, collectio tubes were stored horizotally for 30 mi, 2 h, 4 h ad 6 h before beig cetrifuged ad aalyzed for routie coagulatio tests. Left plasma uderwet a secod cetrifugatio cycle before beig stored froze i aliquots at -80 C for additioal assays. All routie coagulatio tests i.e. prothrombi time (PT, expressed i secod or as the patiet-to-cotrol ratio) or iteratioal ormalized ratio (INR) i patiets o VKA, activated partial thromboplasti time (aptt, expressed i secod or as the patiet-to-cotrol ratio), fibrioge (i g/l), coagulatio factor V (FV, expressed i iteratioal uit per ml, IU/mL) as well as ati-activated factor X (ati-fxa) activity (i IU/mL) whe applicable, were locally performed i each participatig ceters usig their ow routie procedures (Table 1). I additio, factor VIII activity (FVIII, i IU/mL) was evaluated i oe ceter o samples obtaied from hemophiliac patiets usig a oe-stage clottig assay. Platelet factor 4 (PF4) was evaluated usig a ELISA (Asserachrom PF4, Stago, Asières, Frace), accordig to the recommedatios of the maufacturer o the ELx800/KC4 microplate reader (BioTek Istrumets-Fischer, Illkirch, Frace). Test results were expressed i IU/mL. Statistical aalysis Icomplete doublets/quadruplets of tubes, hemolyzed or coagulated samples ad data obtaied without followig the protocol

3 538 P. Toulo et al. / Thrombosis Research 126 (2010) Table 1 Reagets ad istrumets used i the differet ceters (A, B, C, ad D) for the measuremet of prothrombi time (PT), activated partial thromboplasti time (aptt), fibrioge (Fg), clottig factor V (FV) ad factor VIII (FVIII) ad Ati-FXa activity. The complete list of reagets, calibrators ad aalyzers maufacturers used i the study is reported i Appedix A. Ceter Test Reagets Calibrator Aalyzer A PT/INR HemosIL Recombiplasti (IL) HemosIL Calibratio plasma (IL) ACL TOP (IL) aptt HemosIL TCA SP (IL) N. appl Fg HemosIL Fibrioge Clauss (IL) HemosIL Calibratio plasma (IL) FV STA-Deficiet V (Stago) HemosIL Calibratio plasma (IL) Ati-FXa HemosIL Hepari (IL) Hepari Calibrator Global (IL) B PT/INR Neoplastie CI 10 (Stago) STA-Uicalibrator (Stago) STA-R (Stago) aptt PTT Automate (Stago) N. appl Fg Fibrioge (Stago) STA-Uicalibrator (Stago) FV STA Deficiet V (Stago) STA-Uicalibrator (Stago) Ati-FXa Rotachrom Hepari (Stago) STA-Hepaorm H (Stago) C PT/INR Neoplastie CI 10 (Stago) Normal Referece Plasma (Cryopep) STA-R (Stago) HemosIL Recombiplasti (IL)* Normal Referece Plasma (Cryopep)* ACL TOP (IL)* aptt Automated APTT (Triity) N. appl Fg Fibriquick (Triity) Normal Referece Plasma (Cryopep) Thrombi (Siemes)* Normal Referece Plasma (Cryopep)* FV STA Deficiet V (Stago) Normal Referece Plasma (Cryopep) FVIII Factor VIII Deficiet (Cryopep) Normal Referece Plasma (Cryopep) Ati-FXa Rotachrom Hepari (Stago) Hepari Calibrator (Biopep) Coamatic Hepari (IL)* Hepari Calibrator Global (IL)* D PT/INR HemosIL Recombiplasti (IL) HemosIL Calibratio plasma (IL) ACL TOP (IL) aptt Plateli LS (Triity) N. appl Fg HemosIL Fibrioge Clauss (IL) HemosIL Calibratio plasma (IL) FV FV Depleted plasma (Siemes) HemosIL Calibratio plasma (IL) Ati-FXa BIOPHEN Hepari 6 (Hyphe Biomed) BIOPHEN Hepari Calibrator (Hyphe Biomed) *The aalyzer ad some of the reagets used i the ceter C were ot idetical i the two parts of the study (stars idicate techical coditios i the secod part of the study). N. appl.: o applicable. specificatios were excluded from the study. Sice most of the data were ot ormally distributed, test results were expressed as the media values with their rages. Accordigly, comparisos were performed usig the Wilcoxo siged-raks test for matched samples ad iterdepedece of data was evaluated usig the oparametric Spearma correlatio coefficiet (r). Aalysis of variace (ANOVA) was used to evaluate the time-depedet chage i coagulatio test results, which were expressed as the mea values with their stadard deviatios sice they were ormally distributed. Liear regressio betwee test results obtaied i the differet tubes was calculated accordig to Passig ad Bablock [27].A p value smaller tha 0.05 was cosidered sigificat. I additio, the bias aalysis, evaluated accordig to Blad ad Altma [28], was used to evaluate the cliical agreemet betwee test results obtaied i the differet tubes. The stadard deviatio used to determie the acceptable limit was the oe of the iteral quality cotrol of each parameter [29] that was averaged due to the differet techical coditios i.e for PT ad aptt ratios, 0.15 g/l for fibrioge, 0.05 IU/mL for FV, ad 0.05 IU/mL Table 2 Evolutio over a 6-hours period of time of coagulatio test results i.e. prothrombi time (PT, i secods), activated partial thromboplasti time (aptt, i secods), fibrioge (Fg, i g/l) ad factor V (FV, i IU/mL) measured i partial- ad i full-draw tubes cotaiig M citrate as the aticoagulat solutio, that were obtaied from healthy idividuals ( betwee brackets). For further details, see Patiets, Materials ad Methods part. Test Ceter Tube Delay betwee blood collectio ad cetrifugatio 30 mi 2 h 4 h 6 h PT (sec) A (=10) Full 10.7± ± ± ±0.3 Partial 10.7± ± ± ±0.3 B (=13) Full 12.5± ± ± ±0.5 Partial 12.3± ± ± ±0.5 C (=13) Full 12.9± ± ± ±0.6 Partial 12.9± ± ± ±0.6 aptt (sec) A (=10) Full 29.5± ± ± ±2.1 Partial 29.6± ± ± ±2.4 B (=13) Full 34.2± ± ± ±3.9 Partial 35.0± ± ± ±3.5 C (=13) Full 33.0± ± ± ±4.2 Partial 33.0± ± ± ±4.2 Fg (g/l) A (=10) Full 3.67± ± ± ±0.69 Partial 3.54± ± ± ±0.68 B (=13) Full 2.92± ± ± ±0.47 Partial 2.82± ± ± ±0.43 C (=10) Full 3.27± ± ± ±0.40 Partial 3.17± ± ± ±0.37 FV (IU/mL) A (=13) Full 1.07± ± ± ±0.20 Partial 1.07± ± ± ±0.18 B (=13) Full 0.95± ± ± ±0.21 Partial 0.93± ± ± ±0.19 C (=10) Full 1.23± ± ± ±0.18 Partial 1.18± ± ± ±0.17

4 P. Toulo et al. / Thrombosis Research 126 (2010) Table 3 Coagulatio test results, expressed as the media values with their rages, measured i partial-draw ad full-draw tubes cotaiig M citrate obtaied from utreated patiets (=136), patiets o vitami K-atagoists (VKA, =84), ad patiets o low molecular weight hepari (LMWH, =48) i.e. prothrombi time (PT, expressed as the patiet-to-cotrol ratio), iteratioal ormalized ratio (INR, i patiets o VKA), activated partial thromboplasti time (aptt, expressed as the patiet-to-cotrol ratio), fibrioge (i g/l), factor V ad factor VIII levels (i IU/mL), ad ati-fxa activity (i IU/mL, i patiets o LMWH). Aalytical Bias r Compariso PT (ratio) ( ) 1.11 ( ) b INR ( ) 2.22 ( ) NS (0.66) NA aptt (ratio) ( ) 1.13 ( ) b Fibrioge (g/l) ( ) 4.09 ( ) b Factor V (IU/mL) ( ) 1.12 ( ) NS (0.22) NA Factor VIII (IU/mL) ( ) 0.43 ( ) b Ati-FXa (IU/mL) (LMWH) ( ) 0.26 ( ) b B-A: Blad-Altma aalysis ; NA: ot applicable ; NS: ot sigificat. for ati-fxa activity. These values correspoded to coefficiets of variatio (CV%) below 5% for all parameters except for the ati-fxa activity (8.5%). Results Stability of coagulatio test results over a 6 hours-period Blood samples from a total of 36 healthy idividuals were aalyzed after citrated collectio tubes were stored horizotally for 30 mi, 2 h, 4 h, ad 6 h. Evolutio of test results was ot sigificatly differet i partial- ad i full-draw tubes cotaiig M citrate as the aticoagulat solutio, over the 6-hours period of time (ANOVA) (Table 2). Compariso of test results i partial- vs. full-draw citrate collectio tubes I utreated patiets (=136), icludig 11 with hemophilia A, as well as i patiets treated with VKA (=84) or LMWH (=48), FV was ot sigificatly differet whe evaluated i partial- ad i fulldraw s, ad the same applied for INR i those patiets o VKA (Table 3). I cotrast, aalytical compariso of test results obtaied i the two tubes demostrated sigificat differeces for PT, aptt, Fibrioge, FVIII ad ati-fxa, but the differece was ot relevat, as the bias betwee test results, calculated accordig to Blad ad Altma, was below oe SD of the iteral quality cotrol for the studied parameter. I all cases, tests results obtaied i the two tubes were highly sigificatly correlated with correlatio coefficiets (r) i the rage from to I patiets o UFH (=89), PT, fibrioge ad FV were ot sigificatly differet whe evaluated i partial- ad i full-draw-tubes whereas aptt was sigificatly shorter ad ati-fxa activity was sigificatly lower i the partial-draw tubes (Table 4). That sigificat discrepacy was observed i the differet techical coditios at each participatig ceters (ot show). However, aptt ad ati-fxa results obtaied i the two tubes were sigificatly correlated with r= ad r=+0.968, respectively (pb i both cases). Whe evaluated accordig to Blad ad Altma, the mea bias betwee tests results obtaied i partial- ad i full-draw tubes was sec for aptt (Fig. 1, pael A) ad IU/mL for ati-fxa activity (Fig. 1, paelb)with sigificat treds toward higher bias with higher aptt ratio ad ati- FXa activity. As a example, the bias betwee ati-fxa activities evaluated i partial- vs. full-draw tubes was IU/mL for ati-fxa above 0.20 IU/mL ad IU/mL for ati-fxa above 0.30 IU/mL. If IU/mL was used as the therapeutic rage for ati-fxa, moitorig hepari therapy usig partial-draw tubes would have led to a uderestimatio of aticoagulatio i 12 of the 84 evaluated patiets o high dose UFH (14.3%). Actually, five patiets had a ati- FXa withi the therapeutic rage whe evaluated i partial-draw tubes whereas they were over-aticoagulated i full-draw-tubes (0.47 vs. 0.75; 0.53 vs. 0.90; 0.63 vs. 0.71; 0.62 vs. 0.71; ad 0.65 vs. 0.72). Seve other patiets had a ati-fxa below the therapeutic rage whe evaluated i partial-draw tubes whereas they were correctly aticoagulated i full-draw-tubes (0.19 vs. 0.35; 0.19 vs. 0.30; 0.29 vs. 0.44; 0.24 vs. 0.39; 0.24 vs. 0.39). Usig either types of tube would ot have iduced ay chage i the hepari dosage i the other 72 patiets o UFH. Platelet factor 4 assay Plasma PF4 levels, evaluated 30 mi after blood collectio from 10 healthy voluteers, were sigificatly higher i partial- tha i fulldraw s [media value=392 IU/mL (rage: ) vs. 177 (rage: ) respectively, pb0.005]. This two-fold differece remaied uchaged whe PF4 was evaluated i the plasma from tubes cetrifuged 2 h, 4 h ad 6 h after blood collectio. These results suggest icreased platelet activatio i the partial-draw tubes compared to full-draw tubes. Table 4 Coagulatio test results, expressed as the media values with their rages, measured i partial-draw ad full-draw tubes cotaiig M citrate obtaied from 89 patiets o ufractioated hepari i.e. prothrombi time (PT, expressed as the patiet-to-cotrol ratio), activated partial thromboplasti time (aptt, expressed as the patiet-to-cotrol ratio), fibrioge (i g/l), factor V levels (i IU/mL), ad ati-fxa activity (i IU/mL). Aalytical Bias r Compariso PT (ratio) ( ) 1.14 ( ) NS (0.18) NA aptt (ratio) ( ) 2.11 ( ) b Fibrioge (g/l) ( ) 4.29 ( ) NS (0.33) NA Factor V (IU/mL) ( ) 1.25 ( ) NS (0.18) NA Ati-FXa (IU/mL) ( ) 0.39 ( ) b B-A: Blad-Altma aalysis ; NA: ot applicable ; NS: ot sigificat.

5 540 P. Toulo et al. / Thrombosis Research 126 (2010) A aptt (ratio) partial-draw mius full-draw tubes B Ati-FXa (IU/mL) partial-draw mius full-draw s 3,5 3,0 2,5 2,0 1,5 1,0 0,5-0,5-1,0-1,5 0,56 Mea -0, SD -1,27-2,0-2,5-3,0-3,5 0,5 1,5 2,5 3,5 4,5 5,5 6,5 7,5 aptt (ratio) Average of partial-draw ad full-draw s 0,4 0,3 0,2 0,1-0,1-0,2-0,3-0,4 0,2 0,4 0,6 0,8 1,0 1,2 1,4 Ati-FXa (IU/mL) average of partial-draw ad full-draw s Compariso of partial-draw CTAD vs. full-draw s 7 Mea SD -0,20 Fig. 1. Compariso, performed accordig to Blad ad Altma, of activated partial thromboplasti time (aptt, expressed as the patiet-to-cotrol ratio, pael A) ad Ati- FXa activity (i IU/mL, pael B) evaluated i partial- ad full-draw tubes cotaiig M citrate as the aticoagulat solutio, collected from 89 patiets o ufractioated hepari. I the 106 utreated patiets, icludig 8 with hemophilia A, FV ad FVIII levels were ot sigificatly differet i the two tubes whereas the differece was sigificat for PT, aptt, ad fibrioge (Table 5). However, the differece was ot relevat as the bias betwee test results, evaluated accordig to Blad ad Altma, was below oe SD of the iteral quality cotrol for these parameters. I patiets o UFH (=98), aptt was ot sigificatly differet i the two tubes (Fig. 2, paela),adthesameappliedtofibrioge ad FV levels. Aalytical compariso showed sigificatly higher ati-fxa activity i partial-draw CTAD tubes tha i full-draw s, as expected from the lower platelet activatio i the CTAD tubes. That sigificat differece was observed i all four ceters, ad the same A aptt (ratio) partial-draw CTAD mius full-draw s B Ati-FXa (IU/mL) partial-draw CTAD mius full-draw s 3,5 3,0 2,5 2,0 1,5 1,0 0,5 Mea 0, SD -0,5-0,38-1,0-1,5-2,0-2,5-3,0-3,5 0,5 1,5 2,5 3,5 4,5 5,5 6,5 7,5 0,4 0,3 0,2 0,1-0,1-0,2-0,3 aptt (ratio) Average partial-draw CTAD ad full-draw s 9 Mea SD -5-0,4 0,2 0,4 0,6 0,8 1,0 1,2 1,4 Ati-FXa (IU/mL) Average of partial-draw CTAD ad full-draw s Fig. 2. Compariso, performed accordig to Blad-Altma, of activated partial thromboplasti time (aptt, expressed as the patiet-to-cotrol ratio, pael A) ad Ati-FXa activity (i IU/mL, pael B) evaluated i partial-draw tubes cotaiig CTAD ad i full-draw tubes cotaiig M citrate as the aticoagulat solutio, collected from 98 patiets o ufractioated hepari. applied after havig pooled data obtaied from all ceters [media value=0.21 IU/mL (rage: ) vs (rage: ), pb0.0001], as show i Table 6. However, the differece was ot relevat with a mea bias betwee test results obtaied i the two tubes as low as IU/mL, idetical throughout the measurig rage (Fig. 2, pael B). If IU/mL was used as the therapeutic rage for ati-fxa, discrepat test results were foud i oly 6 patiets (6.5%), all of these discrepat results beig observed for borderlie ati-fxa values. Actually, ati-fxa was foud to be withi the therapeutic rage i partial-draw CTAD tube ad below the therapeutic rage i full-draw i five cases i.e vs. 0.25; 0.33 vs. 0.28; 0.31 vs. 0.29; 0.36 vs ad 0.38 vs The opposite was observed i a sigle case: 0.28 vs Usig either types of tube would ot have iduced ay Table 5 Coagulatio test results, expressed as the media values with their rages, measured i partial-draw CTAD tubes ad i full-draw s obtaied from 106 utreated patiets i.e. prothrombi time (PT, expressed as the patiet-to-cotrol ratio), activated partial thromboplasti time (aptt, expressed as the patiet-to-cotrol ratio), fibrioge (i g/l), factor V ad factor VIII levels (i IU/mL). CTAD tube Aalytical Bias r Compariso PT (ratio) ( ) 1.04 ( ) b aptt (ratio) ( ) 1.05 ( ) b Fibrioge (g/l) ( ) 3.39 ( ) b Factor V (IU/mL) ( ) 1.14 ( ) NS (0.23) NA Factor VIII (IU/mL) ( ) 1.04 ( ) NS (0.38) NA 0.976

6 P. Toulo et al. / Thrombosis Research 126 (2010) Table 6 Coagulatio test results, expressed as the media values with their rages, measured i partial-draw CTAD tubes ad i full-draw s obtaied from patiets o ufractioated hepari (=98) i.e. prothrombi time (PT, expressed as the patiet-to-cotrol ratio), activated partial thromboplasti time (aptt, expressed as the patiet-tocotrol ratio), fibrioge (i g/l), factor V levels (i IU/mL), ad ati-fxa activity (i IU/mL). CTAD tube Aalytical Bias r Compariso PT (ratio) ( ) 1.14 ( ) b aptt (ratio) ( ) 1.64 ( ) NS (0.35) NA Fibrioge (g/l) ( ) 5.01 ( ) NS (0.17) NA Factor V (IU/mL) ( ) 1.12 ( ) NS (0.13) NA Ati-FXa (IU/mL) ( ) 0.20 ( ) b chage i the hepari dosage i the other patiets o UFH (93.5% of the patiets o UFH). I additio, aptt ad ati-fxa results obtaied i the two tubes were highly sigificatly correlated [r= (95%CI: ) ad r= (95%CI: ) respectively; pb i both cases] with regressio lies close to idetity (Fig. 3). Coclusios We here reported that the partial-draw collectio tube VeoSafe cotaiig M sodium citrate was suitable for routie hemostasis testig i utreated patiets as well as i patiets o VKA or o the Fig. 3. Correlatio, performed accordig to Passig-Bablock, of activated partial thromboplasti time (aptt, expressed as the patiet-to-cotrol ratio, pael A) ad Ati- FXa activity (i IU/mL, pael B) evaluated i partial-draw tubes cotaiig CTAD ad i full-draw tubes cotaiig M citrate as the aticoagulat solutio, collected from 98 patiets o ufractioated hepari. LMWH derivative eoxapari. Routie coagulatio test results obtaied i those partial- ad full-draw s were either ot sigificatly differet or with a differece remaiig withi acceptable limits (Blad- Altma). However, a sigificat uderestimatio of aticoagulatio was demostrated i patiets o UFH, i all of the four participatig ceters, whe blood was collected i partial-draw tubes. These results are cogruet with those reported i the literature [21,22]. However, it is ot possible to rule out that such a eutralizig effect could be observed with LMWH's other tha eoxapari, especially with compouds with loger polysaccharide chais such as tizapari. Actually, i our study, oly three patiets were treated with that LMWH, makig impossible ay statistical aalysis. This discrepacy appeared to be due to sigificatly higher platelet activatio i partial-draw tha i fulldraw tubes, as suggested by sigificatly higher PF4 levels measured i partial-draw tubes. Actually PF4 is a chemokie of CXC subfamily that is stored i platelet alpha-graules ad released i the plasma after platelet activatio [29]. Beside its mai patho-physiological role i iflammatio process by ehacig chemotaxis, release of histamie by moocytes, adhesio of eosiophiles, abrogatio of moocytes, apotosis, or differetiatio of moocytes ito macrophages [30], released PF4 is able to bid with high affiity hepari as well as other polysaccharides ad egatively charged compouds ad so to eutralize biological effects of these molecules [31]. To further support that hypothesis, blood was collected from patiets o UFH i partial-draw tubes cotaiig CTAD, a mixture of aticoagulat ad atiplatelet agets, istead of citrate, as the aticoagulat solutio. As the results, aptt was ot sigificatly differet i partial-draw CTAD tubes ad i full-draw s, whereas the sigificat aalytical differece was ot foud to be relevat for ati-fxa activity whe data were compared accordig to Blad ad Altma. Altogether, these results suggest that collectig blood ito partialdraw s resulted i icreased platelet activatio. However, the mechaism resposible for that activatio remais questioable e.g. icreased blood-to-iside tube materials or blood-to-air iteractios, lower vacuum ad deserves to be further ivestigated. I coclusio, our study suggests that samples collected ito partialdraw s allow accurate routie coagulatio testig i all patiets but those requirig UFH assessmet. I those patiets o UFH, their use could lead to a sigificat uderestimatio of aticoagulatio due to the hepari-eutralizig effect of PF4 released from activated platelets. I such cases, partial-draw tubes cotaiig CTAD could be validly used to moitor hepari therapy, as well as to perform routie coagulatio testig. As similar results were also reported with aother brad of partial-draw citrate collectio tubes from aother maufacturer [32], cliical laboratory staff ad cliicias must draw special attetio to the type of collectio tubes used for moitorig treatmets with UFH, especially i pediatric patiets ad avoid the use of citrate partial-draw tube for that purpose. Coflict of iterest statemet Noe (for all co-authors).

7 542 P. Toulo et al. / Thrombosis Research 126 (2010) Ackowledgmets The authors wish to thak the Hemostasis Laboratory techicias ad the urse staff from Cetre Hospitalier R. Ballager (Aulay sous Bois, Frace), Hôpital Simoe Veil (Eauboe-Motmorecy, Frace), CHU Hôtel-Dieu (Nates, Frace), ad Hôpital Sait-Roch ad Hôpital Cimiez (Nice, Frace) for their skillful assistace durig the realizatio of the study. Appedix A. List of the reagets/aalyzers maufacturers listed i the text (alphabetical order): BioPep/CryoPep, Motpellier, Frace Hyphe BioMed, Neuville-sur-Oise, Frace Istrumetatio Laboratory (IL), Bedford, MA, USA Siemes, Marburg, Germay Stago, Asières, Frace Triity Biotech, Bray, Co Wicklow, Irelad Refereces [1] Ridyard J, Bhavai M, Seal LH. Laboratory cotrol of oral aticoagulat therapy: preservatio of prothrombi time specimes usig a polypropylee collectio system. Cli Lab Haematol 1998;20: [2] D'Agelo G, Villa C. 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