The combined use of neutrophil gelatinase-associated lipocalin and brain natriuretic peptide improves risk stratification in pediatric cardiac surgery

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1 Clin Chem Lb Med 212;5(11): by Wlter de Gruyter Berlin Boston. DOI /cclm The combined use of neutrophil geltinse-ssocited lipoclin nd brin ntriuretic peptide improves risk strtifiction in peditric crdic surgery Mssimilino Cntinotti1, Simon Storti 1, Vlentin Lorenzoni2, Luigi Arcieri1, Riccrdo Moschetti1, Bruno Murzi1, Isbell Spdoni 1, Cludio Pssino 1,3 nd Aldo Clerico1,3, * 1 Fondzione Toscn G. Monsterio, Mss, Itly 2 CNR Institute of Clinicl Physiology, Pis, Itly 3 Scuol Superiore Snt Ann, Pis, Itly Abstrct Bckground : The im of this study is to test the hypothesis whether the combined use of crdio-specific biomrker, the brin ntriuretic peptide (BNP) nd mrker of erly renl dmge, the ssy of urinry neutrophil geltinse-ssocited lipoclin (ungal), my improve risk strtifiction in peditric crdic surgery. Methods: We prospectively enrolled 135 children [medin ge 7 (interqurtile rnge 1 49) months] undergoing to crdic surgery for congenitl hert disese. All biomrkers were evluted pre- nd post-opertively t different times fter crdiopulmonry-bypss (CPB): ungal t 2, 6 nd 12 h; BNP t 12 nd 36 h; serum cretinine t 2, 6, 12, nd 36 h. Primry endpoints were development of cute kidney injury (AKI) (defined s 1.5 serum cretinine increse) nd intubtion time. Results: AKI occurred in 39 % of ptients (65 % neontes nd 32 % older children, p =.4). The pek of ungal vlues occurred more frequently t 2 h. ungal vlues t 2 h [medin 28.2 (interqurtile rnge ) ng/l] hd good dignostic ccurcy for erly dignosis of AKI with n AUC (re under the curve) ROC (receiver operting chrcteristic) curve of.85 (SE.34). Using multivrible logistic regression nlysis, development of AKI ws significntly ssocited with ungal vlues t 2 h fter CPB [OR = 1.88 ( , p =.1)], together with the CPB time nd Aristotle score, s n index of complexity of the surgicl procedure, while pre-opertive BNP vlues were not. Furthermore, ungal nd pre-opertive BNP vlues (together with Aristotle score) were significntly ssocited with dverse outcome (longer intubtion time nd mortlity). *Corresponding uthor: Prof. Aldo Clerico, MD, Lbortory of Crdiovsculr Endocrinology nd Cell Biology, Fondzione CNR Regione Toscn G. Monsterio, Scuol Superiore Snt Ann, Vi Trieste 41, Pis, Itly E-mil: clerico@ifc.cnr.it Received Februry 27, 212; ccepted April 23, 212; previously published online My 13, 212 Conclusions: Pre-opertive BNP nd ungal vlues fter surgery (together with the Aristotle score) were independently ssocited with more severe course nd worse outcome in children undergoing crdic surgery for congenitl hert disese. Keywords: brin ntriuretic peptide (BNP); children; neutrophil geltinse-ssocited lipoclin (NGAL); peditric crdic surgery; renl dmge. Introduction Acute kidney injury (AKI) is common nd severe compliction in children undergoing crdic surgery for congenitl hert disese (1 9), with importnt consequences in terms of cost, morbidity nd mortlity. Unfortuntely, dignosis nd mngement of AKI in this clinicl setting re t present indequte (1 14). This is t lest prtly due to the incomplete understnding of the pthophysiology of renl dmge fter peditric crdic surgery, which limits dignostic nd therpeutic pproches. Recently, AKI syndromes, including those complicting crdic surgery, re considered to be consequence of complex interctions between crdic nd renl injury, so clled crdiorenl syndromes (1 12). Previous studies on crdiorenl syndromes were focused on the renl ischemi-reperfusion dmge cused by the systemic inflmmtory response cused by crdio-pulmonry bypss, the hemodynmic instbility fter surgery nd/or the use of potentilly nephrotoxic drugs (such s loop diuretics) (1, 11). Although disesed hert hs severl negtive effects on kidney function, t the sme time, renl filure cn significntly ffect crdic function (12). Thus, direct nd indirect effects of hert or kidney dysfunction cn thus, initite nd perpetute the disorder of both orgns through complex combintion of neurohormonl feedbck mechnisms (12). The interest in biomrkers of crdic nd renl dmge in children undergoing crdic surgery for congenitl hert disese hs progressively incresed during the lst few yers (1 9). The clinicl relevnce of brin ntriuretic peptide (BNP) in peditric crdic surgery hs been cknowledged in the lst few yers (1), lthough extensive studies re still lcking. The clinicl relevnce of novel biomrkers more specific for AKI syndromes, however, hve been recently evluted in dult nd peditric crdic surgery (4 9), including the neutrophil geltinse-ssocited lipoclin (NGAL), interleukin-18 (IL-18), kidney injury molecule-1 (KIM-1) nd liver-type ftty cid binding protein (L-FABP) (3, 12). Authenticted Downlod Dte 11/2/12 9:46 AM

2 21 Cntinotti et l.: Combined NGAL nd BNP ssy in peditric crdic surgery The dignosis of AKI is currently bsed on seril serum cretinine mesurements, which present some limittions leding to consistent dely in the dignostic process of AKI nd filure in the identifiction of sub-clinicl dmge (2, 3, 15 18). It is theoreticlly conceivble tht the use of biomrker specific for the renl tubulr dmge my improve the dignosis nd mngement of AKI syndromes (3, 18). Among the novel new biomrkers recently suggested for AKI syndromes, the recent guidelines from the Acute Dilysis Qulity Inititive (ADQI) (17) reported tht NGAL could be integrted into clinicl prctice in the ner future. The few studies conducted in ptients with congenitl hert defects undergoing crdic surgery, however, reported conflicting results (4 9). In prticulr, lrge differences in dignostic ccurcy of NGAL in detecting AKI were found (18, 19). These discrepncies between studies my be ttributble to differences in experimentl protocols (such s the choice of different smpling times or clinicl conditions of the enrolled ptients) nd criteri dopted for the dignosis of AKI (15, 2). None of the previous studies moreover, on AKI fter peditric crdic surgery (4 9) investigted the crdiorenl syndrome s whole, only evluting the clinicl performnce of some biomrkers of renl dmge. The min im of the present study is to test the hypothesis whether the combined use of crdio-specific biomrker, the BNP nd mrker of erly renl dmge, the ssy of urinry neutrophil geltinse-ssocited lipoclin (ungal), my improve risk strtifiction in peditric crdic surgery. Another im of this study is to evlute whether the mesurement of ungal nd plsm BNP my significntly nd independently improve the dignostic ccurcy of AKI in these ptients. Mterils nd methods Experimentl protocol Between December 21 nd November 211, we prospectively enrolled 135 children undergoing crdic surgery for correction/pllition of congenitl hert defects t the Peditric Crdic Surgery Division, Hert Hospitl, Fondzione G. Monsterio, Mss, Itly. Ptients were excluded if they hd history of prior renl trnsplnttion or dilysis requirements. The study ws pproved by the Locl Ethic Committee. Informed consent ws obtined from prents of ll the children enrolled in the study. Biomrker mesurements Lbortory investigtors were blinded to clinicl outcomes. The concentrtions of NGAL in urine specimens (ungal) were mesured by fully utomted immunossy using the ARCHITECT pltform (Abbott Dignostics Lbortories, Abbott Prk, IL, USA). Urine smples were collected before nd t 2, 6 nd 12 h fter termintion of crdiopulmonry bypss nd stored in liquots t 8 C. Becuse cretinine ws lso mesured in the sme urine smple, it ws possible to clculte the rtio between the NGAL nd cretinine concentrtions. Cretinine in serum nd urine smples ws mesured by n enzymtic method (21) with the UniCel DxC 6 pltform (CR-E, REF A6298, Beckm Coulter Beckmn Coulter, Inc., Fullerton, CA, USA). Blood nd urine smples for the mesurement of cretinine were collected pre-opertively nd lso post-opertively t 2, 6, 12, nd 36 h fter termintion of crdio-pulmonry bypss (CPB). Urine smples were lso collected on the wrd until hospitl dischrge, dependent upon the ptients clinicl condition nd tretment. Blood smples for BNP mesurement were collected preopertively nd then t 12 h fter opertion, using only blood smples tken for clinicl necessity (t 7:3 m). No dditionl smples were withdrwn. Plsm BNP ws mesured using the fully utomted Access pltform (Trige BNP regents, Access Immunossy Systems, REF 982; Beckmn Coulter, Inc., Fullerton, CA, USA). The nlyticl chrcteristics nd performnce of the Access Immunossy method used in this study for mesurement of BNP hd been previously evluted in our lbortory (22, 23). Arteril blood gses nd rteril lctte were mesured with fully utomted ssy (ABL 7 series Rdiometer Copenhgen) t the sme points of those of serum cretinine. Outcome nd vribles definitions The primry outcomes of the study were the presence of n AKI syndrome nd the intubtion time fter crdic surgery. The criteri for the presence of AKI were n increse of 1.5-fold or greter of plsm cretinine from pre-opertive bseline levels ccording to the RIFLE clssifiction modified for peditric ptients (15, 16). Surgicl procedures were clssified ccording to the Aristotle score, s index of complexity of surgicl procedure (24). Clinicl prmeters, including ge, gender, weight, height, body surfce re, nd durtion of crdiopulmonry bypss were collected for every ptient. Adverse events were recorded up to 3 dys fter crdic surgery ccording to Portmn et l. (25). Surgicl nd clinicl mngement The pre-opertive nesthesi mngement, intr-opertive bypss strtegy, nd subsequent mngement in the intensive cre unit (ICU) followed stndrd institutionl prctice. Non-iodinted topicl ntiseptics were used for every ptient. A stndrd technique ws used to institute CPB (roller pump, disposble membrne oxygentor nd rteril filter) nd involved bicvl dringe nd scending ort perfusion. Different myocrdil protection (nterogrde cold crystlloid crdioplegi or with cold blood crdioplegi) nd degrees of body temperture were used (rnging from 35 C to 19 C) depending on the surgicl strtegy. In the post-opertive period, hemodynmic mngement ws conducted using epinephrine (.5.15 ng/kg/min), milrinone (.5.75 ng/kg/min), dopmine (5 2 ng/kg/min), nd nordrenline (.5.5 ng/kg/min). Intrvsculr volume expnsion ws conducted ccording to the ttending physicin nd consisted of 2 % humn lbumin or freshfrozen plsm. Diuretics usully consisted of furosemide (1 1 mg/kg/dy). Echocrdiogrms were performed before nd fter crdic surgery (usully between 12 nd 24 h fter opertion) nd ejection frction ws derived ccording to current guidelines (26). Sttisticl nlysis Dt were expressed in term of medin (25th 75th percentiles) for continuous vribles nd number of subjects (percentge) for ctegoricl vribles. Comprison between groups were performed by Authenticted Downlod Dte 11/2/12 9:46 AM

3 Cntinotti et l.: Combined NGAL nd BNP ssy in peditric crdic surgery 211 Fisher test, χ 2 -test, independent Student s t-test nd non-prmetric Mnn-Whitney U-test. Spermn s rho ws used to nlyze the reltion between vribles. Biomrker vlues over time were evluted with mixedeffects regression models (MRMs) to properly ccount for correltion mong repeted mesures nd missing vlues. Receiver operting chrcteristic (ROC) curves nd the re under the curve (AUC) were used to ssess the discrimintory bility of ungal. Univrible nd multivrible logistic regression ws used to identify vribles ssocited with AKI. The intubtion time (or extubtion time, TTE) ws studied in term of time to event vrible. To do this, zero time ws considered s the time of intubtion (tube plcement) nd extubtion (tube removl) events were considered until the 15th dy post-surgery. Decesed ptients or those requiring longer thn 15 dys intubtion were censored. The Kpln-Meier method nd log rnk test were used to compre the TTE vlues cross groups. Cox proportionl-hzrd models were used to identify vribles ffecting the TTE. Due to the fct we considered TTE s the event studied in term of time to event nlysis, hzrd rtio (HR) should be interpreted s the indictor of chnce to extubtion with HR lower thn one indicting low risk of TTE (negtive outcome) nd HR higher thn one suggesting higher chnce of extubtion (positive outcome). For both logistic regression nd Cox proportionl-hzrd model vribles with p <.1 t univrible nlysis were considered for multivrible models. Proportionl hzrd ssumption ws checked using the Schoenfeld test nd no significnt deprtures from this ssumption were observed. Logrithmic trnsformtion ws used for vribles which re not normlly distributed. A 2-tiled vlue of p <.5 ws considered sttisticlly significnt. SPSS version 13. (SPSS Inc., Chicgo, IL, USA) ws used for nlysis. Results Clinicl dt Clinicl nd demogrphic chrcteristics s well s the results of lbortory tests of the enrolled ptients in bsl conditions (i.e., pre-opertively) re reported in Tble 1, while the prmeters of outcome divided ccording to the different gesubgroups in Tble 2. AKI occurred in 52 children (39 % ). In prticulr, AKI occurred in 65 % of neontes vs. 32 % of older children (p =.4). On verge, clinicl conditions of neontes were more severe thn those of older children, s demonstrted by higher opertive risk, expressed by the Aristotle score (p =.28), nd post-opertive period chrcterized by longer medin intubtion time (p <.1), length of sty in the ICU (p <.1) nd need for inotropic support (p =.11). Tble 1 Ptients chrcteristics. Chrcteristics, units AKI (n = 52) Non AKI (n = 83) Totl (n = 135) Age, m o nt h s 3 ( 33) 11 (4 6 ) 7 (1 49 ) M le, % 3 (57.7 ) 4 8 (57.8 ) 78 (57.8 ) Weight, kg4.8 ( )8.6 ( )7 ( ) Height, cm 57 ( ) 72.5 ( ) 67.5 ( ) Body surfce re, m ( ) 5.6 ( ) 5. 5 ( ) Glenn, % 2 (3.85) 1 (1.2) 3 (2.24) LRVO, % 4 (4.82) 4 (2.99) Left ventriculr pressure overlod, % 8 (15.38)9 (1.84)17 (12.69) Left ventriculr volume overlod, % 16 (3.77)25 (3.12)41 (3.6) Pllited UH, % 2 (3.85)4 (4.82)6 (4.48) Right ventriculr pressure overlod, % 12 (23.8) 12 (14.46) 24 (17.91) Right ventriculr volume overlod, % 2 (3.85)19 (22.89)21 (15.67) Trnsposition of the gret rteries, % 5 (9.62) 3 (3.61) 8 (5.97) Univentriculr hert, % 5 (9.62) 6 (7.23) 11 (8.21) BNP, ng/l 149 ( )57 ( )86 ( ) Serum cretinine, mg/dl.24 (.19.3).24 (.18.3).24 (.18.3) Urinry cretinine, mg/dl 23.2 ( ) 53.8 ( ) ( ) u-ngal, ng/ml7.65 ( )8.3 ( )8.3 ( ) u-ngal/urinry cretinine rtio 4.3 ( ) 2.2 ( ) 2.8 ( ) CPB time, min 153 ( )98 (65 115) 112 (76 15) Cross-clmp, min 83 (29 122) 47 (27 67) 58 (28 83) Intubtion time, h 48 (12 111) 8 (5 14) 12 (5 61) Inotropic time, h 18 (36 182) 12 (1 6) 36 (12 18) ICU sty, h 18 (36 156)24 (12 36)36 (12 18) Aristotle score 8.37 (2.) 6.58 (2.18) 7.27 (2.28) p <.5 Mnn-Whitney U-test or independent Student s t-test. Congenitl hert diseses hve been divided into min groups ccording to the hemodynmic (23): Biventriculr volume overlod group (trio-ventriculr defects); Left ventriculr pressure overlod group (including ortic stenosis nd ortic corcttion); left ventriculr volume overlod group (ventriculr septl defects, significnt ptent rteril duct, truncus rteriosus): Right ventriculr pressure overlod group (tetrlogy of Fllot, pulmonry stenosis); right ventriculr volume overlod group (tril septl defect, nomlous pulmonry venous dringe). The conversion fctor for cretinine from mg/dl to SI Units is The ungal/urinry cretinine rtio ws clculted by the formul: rtio vlue = (ungal, expressed s ng/ml or μg/l)/(urinry cretinine, expressed s mg/dl) 1. Authenticted Downlod Dte 11/2/12 9:46 AM

4 212 Cntinotti et l.: Combined NGAL nd BNP ssy in peditric crdic surgery Tble 2 Prmeters of outcome divided ccording to the different ge-subgroups. Prmeters, units Neontes (n = 26) Older children (n = 19) p-vlue CPB time, min 137 (11 17) 17 (74 143).71 Cross-clmp, min 49 (6 87) 61 (3 84).236 Intubtion time, h 84 (15 174) 8 (5 18) <.1 Inotropic time, h 18 (33 228) 12 (1 84).11 ICU sty, h 18 (42 225)36 (12 6) <.1 A r i st o t le s c o r e 8.16 ( ) 7. 6 ( 2.13). 2 8 Adverse events, % 5 (19.2)9 (8.3).145 [Type of events (n)] [LCO (2), Redo (2), Deth (1)] [LCO (6), Redo (1), CA (2)] CA, crdic rrest; ICU, intensive cre unit; LCO, low crdic output; Redo, ptients requiring re-opertion. Due to the more severe clinicl conditions s well s the higher number of neontes, the group of ptients with AKI showed lso significntly higher (p =.1) vlues of BNP thn ptients without AKI (Tble 1). NGAL nd BNP levels fter crdic surgery Considering ll ptients s whole group, ungal vlues significntly incresed post-surgery, with pek [4.1 ( ) ng/ml] usully occurring between 2 nd 6 h, being more frequent t 2 h (corresponding to the 64 % of the totl number of cses). In prticulr, ungal vlues t 2 h [28.2 ( ) ng/ml] were significntly higher thn in the bsl condition [8.3 ( ) ng/ml; p <.1)]. After this initil, erly increse progressive decrese of ungal ws observed t 6 nd 12 h [18.4 ( ) ng/ml nd 3.8 ( ) ng/ml, respectively]. It is importnt to note tht the time-courses of ungalvlues were different in ptients with nd without AKI. Indeed, in ptients with AKI the ungal vlues t 2 h were significntly higher thn bsl vlues (Figure 1, prt A.2), while in ptients without AKI these unagl vlues were not significntly different to bsl ones (Figure 1, prt A.1). According to the dignosis of AKI (15, 16), serum cretinine vlues significntly incresed only in ptients with AKI (Figure 1, prt B.2). Serum cretinine vlues progressively incresed fter surgery, reching the pek t 36 6 h A.1 B p=.11 ungal, ng/ml b c Serum cretinine, mg/dl A.2 B.2 ungal, ng/ml b c Serum cretinine, mg/dl h b. Pre-surgery 2 h 6 h 12 h 36 h Figure 1 Behvior of ungal nd serum cretinine vlues. Behvior of ungal (Prt A.1 nd A.2) nd serum cretinine (Prt B.1 nd B.2) levels in non-aki group (upper pnels) nd AKI group (lower pnels) of peditric ptients throughout the study. The results re reported s medin (bold horizontl line) nd interqurtile rnge (box). The p-vlues for difference overll the smples collected t different times pre- nd post-surgery (t 2, 6 nd 12 h, respectively) were lso reported. p-vlue <.5 for differences with pre-surgery levels; b p-vlue <.5 for differences with 2 h levels; c p-vlue <.5 for differences with 6 h levels. Authenticted Downlod Dte 11/2/12 9:46 AM

5 Cntinotti et l.: Combined NGAL nd BNP ssy in peditric crdic surgery 213 in ptients with AKI; s result, the concentrtion pek of ungal occurred significntly erlier thn tht of serum cretinine (p <.1) (Figure 1, prt A.2 nd prt B.2). Urinry cretinine showed fll fter surgery (p <.1) nd n increse lter with pek t h in both ptients with nd without AKI (Figure 2 ). The rtio between the concentrtion of NGAL nd cretinine in urine smples significntly incresed fter surgery (p <.1), peking t 2-h nd decresing subsequently with time course similr to tht of ungal (Figure 2). Considering ll ptients s whole, BNP vlues incresed fter surgery [from 86 ( ) ng/l before surgery to ( ) ng/l t 12 h fter surgery; p <.1], showing similr time-courses in ptients with or without AKI. Dignostic ccurcy of NGAL in the erly dignosis of AKI ungal t 2 h showed good dignostic ccurcy for the dignosis of AKI, with n AUC of.85 (SE.34) with cutoff vlue of ng/ml, sensitivity.784 nd specificity of.815. Dt concerning the nlysis of ROC curves, corresponding to smples, collected t different times throughout the study, re reported in Tble 3. A significnt dignostic ccurcy for the presence of AKI ws lso found for BNP levels 12 h fter surgery, with n AUC of.7 (sensitivity.712 nd specificity.612) with best cut-off vlue of ng/l. Inter-reltionships between biomrker vlues nd clinicl outcomes A significnt negtive correltion ws found between ungal vlues nd body surfce re (BSA) ( ρ =.23, p =.31). All ungal vlues observed fter surgery positively correlted with the severity of the crdic disese ( ρ =.26, p =.4 for ungal 2 h post-surgery), s ssessed by pre-surgery BNP vlues, s well s with the indictors of surgery complexity, s ssessed by the Aristotle score ( ρ =.28, p =.1 for ungal 2 h post-surgery) nd CPB-time ( ρ =.62, p <.1 for ungal 2 h post-surgery). Highly significnt positive correltions were lso found between ungal vlues fter surgery nd prmeters of outcomes, such s intubtion durtion ( ρ =.43, p <.1 for ungal 2 h post-surgery) nd time spent in the ICU ( ρ =.46, p <.1 for ungal 2 h post-surgery). Pre-surgery BNP vlues correlted negtively with BSA nd ge ( ρ =.56, p <.1 nd ρ =.51, p <.1, respectively), nd positively with intubtion durtion ( ρ =.44, p <.1), A.1 5 B.1 1 ungal/ur. cretinine b b c Urinry cretinine, mg/dl b b A.2 ungal/ur. cretinine b b c B.2 Urinry cretinine, mg/dl Figure 2 Behvior of ungal/urinry cretinine rtio nd urinry cretinine vlues. Behvior of ungal/urinry cretinine rtio (Prt A.1 nd A.2) nd urinry cretinine (Prt B.1 nd B.2) levels in non-aki group (upper pnels) nd AKI group (lower pnels) of peditric ptients throughout the study. The results re reported s medin (bold horizontl line) nd interqurtile rnge (box). The p-vlues for difference overll the smples collected t different times pre- nd post-surgery (t 2, 6 nd 12 h, respectively) were lso reported. p-vlue <.5 for differences with pre-surgery levels; b p-vlue <.5 for differences with 2 h levels; cp-vlue <.5 for differences with 6 h levels. The ungal/urinry cretinine rtio ws clculted by the formul: rtio vlue = (ungal, expressed s ng/ml or μg/l)/(urinry cretinine, expressed s mg/dl) 1. Authenticted Downlod Dte 11/2/12 9:46 AM

6 214 Cntinotti et l.: Combined NGAL nd BNP ssy in peditric crdic surgery Tble 3 Dt of ROC nlysis. The vlues of best cut-off vlue, AUC, sensitivity nd specificity (clculted t the best cut-off) re reported. Dt group ungal, ng/ml AUC (SE) Sensitivity Specificity 2 h post-surgery (.34) h post-surgery (.36) h post-surgery (.42) Pek mong 12 h post-surgery (.36) Pek within 12 h fter surgery: the highest vlue mesured within 12 h fter surgery. length of sty in the ICU ( ρ =.42, p <.1), nd Aristotle score ( ρ =.24, p =.7). BNP vlues t 12 h fter surgery were lso significntly correlted with 2 h ungal ( ρ =.321, p <.1), thus suggesting link between crdiovsculr nd renl syndrome. Furthermore, BNP vlues t 12 h fter surgery correlted with rteril lctte ( ρ =.44, p <.1), left ventriculr ejection frction ( ρ =.3, p =.7) nd rteril blood ph ( ρ =.4, p =.2) t 12 h fter surgery. Logistic regression Using univrible logistic regression, ll ungal nd NGAL/cretinine rtio vlues fter surgery were found to be significntly ssocited with dignosis of AKI, differently from the pre-opertive vlues. The univrible nlysis lso indicted tht BSA, Aristotle score, CPB time, nd BNP were significntly ssocited with the dignosis of AKI (Tble 4 ). Using multivrite nlysis, the ungal vlues t 2 h postsurgery resulted independently ssocited with dignosis of AKI, together with the CPB time nd the Aristotle score (Tble 4). Univrible nd multivrible Cox regression models Univrible Cox models showed tht higher Aristotle score nd CPB time, but lower ge nd BSA, were significntly ssocited with n incresed risk of longer intubtion times (Tble 5 ). Lower pre-surgery urinry cretinine, higher preopertive nd 12-h BNP nd higher 2-h nd pek ungal vlues moreover, were significnt predictor of longer intubtion times. At multivrible nlysis, Aristotle score, bsl BNP nd ungal t 2 h post-surgery remined the only significnt predictors of intubtion time (Tble 5). Discussion The principl im of the present study ws to evlute whether the combined use of crdio-specific biomrker, such s BNP (27), ssocited with mrker of erly renl dmge, such s ungal (3, 12), my provide better risk strtifiction in peditric ptients with congenitl hert defects undergoing crdic surgery. Another im of this study ws to evlute whether the mesurement of ungal nd plsm BNP my Tble 4 Univrible models Logistic regression models for AKI. Vrible, units OR (CI 95 % ) p-vlue Femle vs. mle 1.1 (.5 2.3).987 Body surfce re, m 2.56 (.36.86).8 Older children vs. neontes.25 (.1.62).3 ungal pre-surgery, ng/ml 1.7 ( ).677 ungal 2 h, ng/ml 2.48 ( ) <.1 ungal 6 h, ng/ml 2.4 ( ) <.1 ungal 12 h, ng/ml 2.5 ( ) <.1 ungal pek, ng/ml 2.85 ( ) <.1 ungal/urinry cretinine 1.29 ( ).81 rtio pre-surgery ungal/urinry cretinine 1.87 ( ) <.1 rtio 2 h ungal/urinry cretinine 1.79 ( ) <.1 rtio 6 h ungal/urinry cretinine 1.85 ( ) <.1 rtio 12 h BNP pre-surgery, ng/l 1.44 ( ).2 BNP 12 h, ng/l 2.18 ( ) <.1 CPB time, h 3.57 ( ) <.1 Cross-clmp, h 3.66 ( ) <.1 Intubtion time, dys 1.17 ( ).1 ICU sty, dys 1.2 ( ).2 Aristotle score 1.55 ( ) <.1 Multivrible model AUC (SE) =.858 (.4) ungal 2 h, ng/ml 1.88 ( ).1 CPB time, h 1.42 ( ).281 Aristotle score 1.44 ( ).25 ICU, intensive cre unit. Log trnsformed vrible. The ungal/ urinry cretinine rtio ws clculted by the formul: rtio vlue = (ungal, expressed s ng/ml or μg/l)/(urinry cretinine, expressed s mg/dl) 1. significntly nd independently improve the dignostic ccurcy of AKI in these ptients. As fr s the risk strtifiction is concerned, the results of the present study demonstrte tht ungal is strongly nd independently ssocited with some dverse events in peditric ptients with congenitl hert defects undergoing crdic surgery (including the longer intubtion time nd the period of time spent in the ICU) (Tble 5). Our study lso indictes tht the pre-opertive BNP vlues were independently ssocited with more severe outcome in peditric ptients undergoing crdic surgery. The results of our study therefore indicte for the first time tht the combined use of ungal nd BNP cn improve risk strtifiction in peditric ptients with congenitl hert defects undergoing crdic surgery. As fr s the dignosis of AKI is concerned, very recent study reported tht plsm BNP is ble to predict AKI in criticlly ill dult ptients (28). In our clinicl setting, only ungal vlues fter surgery, together with CPB time nd the Aristotle score, resulted in independent predictors of development of AKI syndrome, wheres BNP did not (Tble 4). In prticulr, our results strongly support the Authenticted Downlod Dte 11/2/12 9:46 AM

7 Cntinotti et l.: Combined NGAL nd BNP ssy in peditric crdic surgery 215 Tble 5 Cox models for time to extubtion (TTE). Univrible models Vrible, units HR (CI 95 % ) p-vlue Femle vs. mle 1.25 ( ).229 Body surfce re, m ( ) <.1 Older children vs. neontes 2.49 ( ) <.1 Urinry cretinine pre-surgery, 1.41 ( ).4 mg/dl Urinry cretinine 2 h, mg/dl 1.39 ( ).2 Urinry cretinine pek, mg/dl 1.73 ( ) <.1 Serum cretinine 12 h, mg/dl.61 (.45.84).3 Serum cretinine pek, mg/dl.58 (.4.83).3 ungal 2 h, ng/ml.8 (.72.89) <.1 ungal 6 h, ng/ml.72 (.63.81) <.1 ungal 12 h, ng/ml.73 (.64.83) <.1 ungal pek, ng/ml.74 (.66.83) <.1 ungal/cretinine rtio.77 (.66.9).1 pre-surgery ungal/cretinine rtio 2 h.76 (.69.83) <.1 ungal/cretinine rtio 6 h.7 (.63.78) <.1 ungal/cretinine rtio 12 h.71 (.63.81) <.1 ungal/cretinine rtio pek.74 (.67.82) <.1 BNP pre-surgery, ng/l.8 (.7.91).1 BNP 12 h, ng/l.64 (.52.78) <.1 CPB time, h.76 (.64.9).1 Cross-clmp, h.61 (.46.82).1 ICU sty, dys.4 (.32.51) <.1 Aristotle score.73 (.67.81) <.1 Multivrible model Aristotle score.76 (.68.84) <.1 BNP pre-surgery, ng/ L.82 (.71.94).4 ungal 2 h, ng/ml.84 (.75.94).3 HR < 1 suggested lower chnce of extubtion or conversely longer intubtion time; HR < 1 suggested higher chnce of extubtion or eqully lower intubtion time; ICU, intensive cre unit. Log trnsformed vrible. The ungal/urinry cretinine rtio ws clculted by the formul: rtio vlue = (ungal, expressed s ng/ml or μg/l)/ (urinry cretinine, expressed s mg/dl) 1. hypothesis tht ungal my llow n erly dignosis of AKI in peditric ptients with congenitl hert defects within 6 h fter crdic surgery with good dignostic ccurcy (Tble 3). Tking into ccount tht serum cretinine significntly increses only 36 h fter surgery (Figure 2), our findings suggest tht ungal cn llow correct dignosis in bout 8 % of ptients t lest 1 dy before thn the serum cretinine. Conflicting results re reported in the literture bout the dignostic ccurcy of NGAL s biomrker of AKI. Although the mjor prt of studies (4 7, 9) suggested tht NGAL, mesured in urine or blood smples, is useful biomrker of AKI, other uthors (8) reported only limited clinicl vlue for plsm NGAL, ssyed erly fter crdic surgery in peditric ptients. However, Koch et l. (8) used plsm (rther thn urine) smples nd point-of-cre testing (POCT) method for the ssy of NGAL. This POCT method shows lower nlyticl sensitivity s compred to the immunossy method used in this present study (18, 29). It is theoreticlly conceivble moreover, tht the ssy of circulting levels of NGAL my be less ccurte thn its mesurement in urine (18), even if the results of recent met-nlysis were not ble to confirm this hypothesis (19). Finlly, the independent nd significnt contribution of CPB time on the prediction of the presence of AKI (Tble 4) is lrgely expected when considering tht CPB cuses complex systemic inflmmtory responses tht significntly contribute to severl dverse postopertive complictions, including renl dmge (3, 31). Study strengths nd limittions of the present study This study hs severl strengths. Firstly, we used prospective cohort design nd rigorous protocol to collect specimens. Our study protocol furthermore, which used severl smpling times, ws ble to clerly define the time course of the biomrkers. Finlly, for the first time, we evluted together n erly mrker of renl dmge (i.e., NGAL) nd mrker of crdic function (i.e., BNP) in order to test the hypothesis tht the combined use of these two biomrkers could provide better risk strtifiction in peditric ptients with congenitl hert defects undergoing crdic surgery. This study, however, does lso hve some limittions. Firstly, the totl number of studied ptients is reltively low. It is importnt however, to note tht congenitl hert defects, especilly those with more complex severe disese, re very rre (only bout 1: 1, neontes present this disese) (32). It is consequently, very difficult to enrol very lrge number of ptients with congenitl hert defects in short time spn. Another limittion is tht our study considered outcome mesures limited to the peri-opertive period. Further studies with longer follow-up re needed to evlute the long-term prognostic vlue of ungal. Conclusions The development of renl complictions fter crdic surgery portends significnt morbidity nd mortlity (33). In prticulr, depending on the definition, post-opertive AKI occurs in 3 % 3 % of ptients, with the need of renl replcement therpy in 1 % 5 % (33). The prognosis mong this subgroup of ptients is poor, with n incresed mortlity risk exceeding 6 % compred with the overll mortlity rte of 2 % 8 % fter crdic surgery (33). ungal fter surgery moreover, (but not BNP) is significnt nd independent predictor for developing AKI. The mesurement of ungal lone in prticulr, ws ble to llow correct dignosis of AKI in bout 8 % of ptients t lest 1 dy before tht of serum cretinine. Clinicins could use this gin in time to initite n erlier nd more pproprite tretment. Conflict of interest sttement Authors conflict of interest disclosure: The uthors stted tht there re no conflicts of interest regrding the publiction of this rticle. Reserch support plyed no role in the study design; in the collection, nlysis, nd interprettion of dt; in the writing of the report; or in the decision to submit the report for publiction. Authenticted Downlod Dte 11/2/12 9:46 AM

8 216 Cntinotti et l.: Combined NGAL nd BNP ssy in peditric crdic surgery Reserch funding: Abbott Dignostics in prt supported this study by gifting the lbortory tests for the nlysis of ungal. Employment or ledership: None declred. Honorrium: None declred. References 1. Cntinotti M, Giovnnini S, Murzi B, Clerico A. Dignostic, prognostic nd therpeutic relevnce of B-type ntriuretic peptide ssy in children with congenitl hert diseses [review]. Clin Chem Lb Med 211;49: Hse M, Devrjn P, Hse-Fielitz A, Bellomo R, Cruz DN, Wgener G, et l. The outcome of neutrophil geltinse-ssocited lipoclin-positive subclinicl cute kidney injury: multicenter pooled nlysis of prospective studies. J Am Coll Crdiol 211;57: Devrjn P. Biomrkers for the erly detection of cute kidney injury. Curr Opin Peditric 211;23: Prikh CR, Devrjn P, Zppitelli M, Sint K, Thiessen-Philbrook H, Li S, et l. Postopertive biomrkers predict cute kidney injury nd poor outcomes fter peditric crdic surgery. 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9 Cntinotti et l.: Combined NGAL nd BNP ssy in peditric crdic surgery 217 hemostsis nd oxidtive stress. Eur J Crdiothorc Surg 23;24: Elhi MM, Khn JS, Mtt BM. Deleterious effects of crdiopulmonry bypss in coronry rtery surgery nd scientific interprettion of off-pump s logic. Acute Crd Cre 26;8: Grne E, Olsen MS, Johnsen SP, Hjortdl V, Andersen H Ø, Nissen H, et l. How do we define congenitl hert defects for scientific studies? Congenit Hert Dis 212;7: Huen S, Prikh CR. Predicting cute kidney injury fter crdic surgery: systemtic review. Ann Thorc Surg 212;93: Authenticted Downlod Dte 11/2/12 9:46 AM