Carsin 1997 PCT Quemados

8/2/2019 Carsin 1997 PCT Quemados

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I I : !%~05-41:?9(96)00124-6

Bums Vol. 23r No. 3, pp. X8-224, 19970 1997 Elsevier Science Ltd for ISBI. AII rights reserved

Irinted in Great Britain0305-4179/97 $17.00+0.00

volution and significance of circulatingrocakitonin levels compared with IL-6, TNFot andendotoxin levels early after thermal injury

ervk Carsir?, Marcel Assicot2, Frkdkric FegeP, Olivier RoyI, Isabelle Iennacinol,erve Le Beverl, Pierre Ainaudl and Claude Bohuon2Centre de Traitement des brtiles, H.I.A. Percy, BP 406,92141 Clamart Cedex, France and2Departement de Biologic Clinique, Institut Gustave Roussy, 94805 Villejui f, France

To determine the evolut ion and significance of circulating pro-calcitonin (ProCT), lL-6, T NFc x and endotoxin levels early afterthermal injuy, we performed a prospect ive, single unit , longi-tudinal study. Forty burn pat ients with total body surface area(TBSA) >30 per cent were studied, of whom 33 suffered aninhalation injury. Blood samples were taken on the day of admis-sion, every 4 h during the f irst day and daily during the f irstweek . All pat ients had increased ProCT and lL-6 levels withoutany proven infect ion. Endotoxin and TNF M levels remained ve rylo w or undetectable. P roCT and IL-6 levels correlated well withthe severity of skin burn inju y (respect ively, ~


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Carsin et a%:Evolution and significance of circulating procalcitonin levels-~ 219

Table I. Characterist ics of the populat ion studied. Group 0,pat ients without inhalation injuries; Group 1, pat ients withindoctr inhalation injuries; Group 2, patien ts with outdo orinhalation injuries-- -Group 0 Group 1 Group 2Patients 7 (5M, 2F) 21 (13M, 8F) 12 (8M, 4F)Age (years) 44&12 42*15 41*14UB S 120&65 193&79 193k76T5SA 49*19 59&18 59&20Number of deaths 0 7 4~~ -

accident enabled us to identify three groups ofpatient:Patients without smoke inhalation injury (group0);patients with smoke inhalation injuries after anaccident which occurred indoors (e.g. home fire)(group I.);patients with endoscopic inhalation lesions afteran accident which occurred outdoors (group 2)..

The characteris tics of these three groups are shownin Table I. Excision and grafting were performed on29 of the 40 patients during the first week ofhospitalization.Healthy volunteers served as a control group forIL-6 TNFg and IroCT (respectively, 10, 19 and IO).MethodsBlood collection was carried out immediately uponadm.iss ion to the ward and then every 4 h until >the24th hour (the reference hour being the hour of theaccident). Next, a daily blood collection was carriedout during the first week. The laboratory is locatedwithin the Care Unit, so samples were transportedand processed within a short time ( < 1 h).Determination of ProCT. Blood samples were centri-fuged at 15OOg for 10 min, and the sera were storedat --8X before ana lysis. ProCT was assayed by anultrasensitive sandwich immunoluminometric assay(Brahms, Berlin, Germany). This determination usestwo assays and two monoclonal antibodies: a firstbinding antibody to an epitope located on thekatacalcin and a second tracing antibody in themid-region of calcitonin. This assay, which does notdetect mature calcitonin, has a sensi tivity of approxi-mately 10 pdml. The luminescence is measuredusin.g a Berilux analyser (Behring@, Mannheim,Germany) against a standard range from 120 to61000 pg/ml; samples exceeding the maximum valuewerle diluted before re-ana lysis.Determination of endotoxin. Endotoxin levels weremeasured during the firs t 3 days following the burninju ry, except for 15 patients in whom the determina-tion was continued until day 7. Blood was collectedin apyrogenic sterile tubes (Kabi endotubes) andstomd in ice. After 2OOg centrifugation for 15 min at4C we immediately obtained a plasma r ich in plate-lets, aliquots of which were prepared under laminarair flow, transferred to sterile apyrogenic tubes and

istored at -8PC until use. The assay technique is anlend-point chromogen ic method based on the activa-tion of a limulus amoebocyte lysate (LAL) byendotoxin (Coatest endotoxinm, Biogenic, Mont-pellier, France). In order to avoid activation and theinhibiting effects of plasma on the LAL test, allsamples were diluted (1:lO) in apyrogenic sterilewater, then heated to 75C for 10 min. The sampleswere then coincubated for 30 mm at 37Y with theLAL; the chromogenic substrate was added and after10 min the reaction was stopped with an aqueoussolution of 50 per cent acetic acid. Absorbances wereread in a plate-reader (Uniskan IP, Labsystems, LesUlis, France). The absorbance of a control issubstracted from these absorbances in order to adjustthe samples intrinsic colour development. The endo-toxin concentration is read on a standardizationcurve plotted after addition of endotoxin (Es&en&acdi Olll:B4-1.2 endotoxin unit (EU)=lO O pg) to apool of control plasmas. This mlethod is sensitive to0.06 EU/ml of endotoxin In accordance with themanufacturer, calculation of recoveries of addedendotoxin allowed the validation of these results(range of acceptable recoveries: 50-150 per cent).Determination of IL-6 and tumaur necrosis factor a.Interleukin-6 (IL-6) and tumour necrosis factor a(TNFa) serum levels were measured using a speci-fic enzyme-linked immunosorbent assay (ELISA)designed as a double antibody sandwich assay(Immunotech, Marseille, France). The normal rangeof IL-6 and TNFa levels observed by these assays are< 10 and < 5 pg/ml, respectively.Blood cultures, lactic acid and PaOJFi02 ratio. Bloodcultu res were performed three times a day for eachpatient, using the BacteP system (Becton-Dickinson,Pant-de-Claix, France). Likewise, arterial lactic acidand IaO#iOZ were determined at the time ofhospital admission, using enzymatic assay(Boehr inger Mannheim, Meylan, France) and ananalyser ABL520@ (Radiometer, Copenhagen,Denmark), respectively.

Statistical analysis. The SPS package for Windows(Version 6.1.2) was used for analysis. We first verifiedthe normality of th e distribution of variables usingthe Shapiro -Wilk test. IL-6 and IroCT distributionwere normal after logarithmic transformation. There-fore, statistical analysis was performed using thelogarithm value of these variablesFor ProCT, IL6 and TNF@, initial values (time HO)were average values of the panel of health contro ls.The role of various factors upon the variation ofIL-6, ProCT and TNF!x during ti.me since injury wastested using non-parametric analysis of variance forrepeated measurements (Friedman test). If this wassignificant, the multiple range test was used (Bonfer-roni) to compare the different values.To take into account the UBS score in the analysis ,we used ANOVA with the UBS score as a covariate.


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To compare the lroCT and IL-6 peaks, and theUBS score among survivors and non-survivors, weused the non-parametric Kruskal-Wallis (KW) test.We used the Spearman rank correlation test (Q) totest thiz link between continued variables.Study of endotoxin variation was monitored usinga Yates x-squared test because a few measurementswere performed on health controls; categories aredefined according to the presence (level 0.06 EU/ml)or absence (level ~0.06 EU/ml) of endotoxin in thesample. The rate of 0.06 EU/ml is the sensitiv itybaseline of the kit used.

Serum levels of ProCT, IL-6 and TNF,z during theweek following a burn injuryThe mean values of serum ProCT, IL-6 and TNFxduring the study period are shown in Figure 1, Meanserum ProCT and IL-6 levels increased during thefirst hours following the burn injury and rapidlystabilized. For IroCT, the increase was significantbetween HO and other postburn times and betweenH4 and H7 and other times (Bonferroni test -p < 0.05). Similarly, the increase in IL-6 was significantbetween HO and other times (p < 0.05).In contrast to IL-6 and ProCT, mean TNFti serumlevels after burn injury did not increase significantlyduring the first seven postburn days (Figure 2). Onlyoue of the 40 patients exhibited increased TNFLY evelsduring the early 24 h, with a peak level of 140 pdmlat 8 h.ProCT #and IL-6 serum leve ls, respiratory burns andUBS scoreWhen the threle groups were compared, the IL-6 andProCT concentrations increased following a similarcurve; Ihowever, the levels were different (Figure 2).For ProCT, we observed a lower mean in group 0than in groups 1 and 2 (KW test). For IL-6, weobserved a higher mean in group 1 (burns, inhalationinjury, closed space) than in groups 0 and 2 (KWtest).

HO H4 - H7 H&H9 HI2 1 8 H2 0 H2 4 DZ D3 D4 I ,5 D7T ime p o st b u m

Figure 1. Serial values (means) of serum IroCT (A), IL-6 (0)and TNFx (0) after burn injury for the entire group of 40patients.

I r 1 I I , , , IHO b &H7 H8-HS,2 1 8 Hx l H2 4 D2 D3 D4 D5 D7

T ime po st b u r n

1 II@ i i : : 1HO H4 - H7 H&W H, ,? h i ,6 H2 0 m+ D2 D3 0 4 D5 D7

T ime p ost b u m

Figure 2. Post burn course of ProCT ant! IL-6 serum levels(means) in each of the three groups: grouP 0 (01, group 1(oh group 2 (9

If we focused on the first 24 h, the same differ-ences were found for IroCT and IL-6 among thedifferent groups.If groups 1 and 2 were considered together (burnpatients with inhalation injury), and the concentra-tions of ProCT and IL-6 were compared with group 0(burn patients without inhalation injury), then higherlevels are observed in patients with inhalation injury(KW test - p < 10d6 and p < 0.003) (T&e 11).Were these levels the results of inhalation injuryor severity of burn injury? Introducing the UBS scoreas a covariate in a variance analysis showed that, inthis model, ProCT and IL-6 did not change signifi-cantly between groups with or without smokeinhalation injury, but correlated with the UBS score(p < 0.006 and p < 0.028) (Figures 3 and 4). Clearly, IL-6and ProCT were not reliable markers of inhalationinjury in these studies.Prognostic value of ProCT and IL-6 serum leve lsEleven patients died during hospitalization. Themaximum values of ProCT and IL-6 measured withinTable II. Peak ProCT and IL-6 levels (means k SEM) withinthe f irst 24 h after burn injury in pat ients with and withoutinhalation injury

ProCT kg/m/) IL-6 bg/m0Without inhalation Group 0 2543kll60 1256 & 384

injury (n = 7)With inhalation Groups 32894&11462 6057k2186

injury (n = 33) 1 and 2


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Carsin et al. : Evolut ion and signif icance of circulating procalcitonin levels 22 1

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ee ell

l

: * l *l

ee

0-I 8100 1000 10000 100000 1000000

Pro,CT concentration (pglml)Figure 3 . Relat ionship between ProCT level and UBS score.

Figwe 4. Relat ionship between IL-6 level and UBS score.

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the first 24 h and the UBS score from survivors andnon-survivors are shown in Fi@ue 5.As shown in Table 111, IroCT, IL-6 and UBS wereprognostic of death, UBS being the best prognosticscore, followed by IL-6 and ProCT. These threeprognostic values were used in a logistic regressionmodel using as parameters ProCT and IL-6 peakvalues within the 24 h following injury and UBS(divided into two groups: ~240 and >240). Thepatient with a UBS score of at least 24@ had 23 timesgreater risk of death than patients with lower UBSvalues. This model gave accurate predictions for 85per cent of patients (Tubk IV).IroCT and IL-6 peaks correlated with UBS (respec-tively, p = 0.61; p < 10m6 and p = 0.47; p = 0.002).

0 35 0l el 30 0e

--J-OProCT IL-6 UBS

Figure 5. I e,ak I roCT and IL-6 levels within 24 h of burn and U BS score in survivors (0, n = 29) and non-survivors (0,72 = 11).


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TableII I . Peak ProCT, IL-6 and UBS score (median 2575%) within the f irst 24 h after burn injury in survivor or non-survivor pat ients The dif ferences for IL-6, ProCT and UBS between groups were signif icant according to KW test

ProCT(pglml) IL-6 kdrnl) UB SSurvivors (f~ = 21)Non-survivors (n = I?)KW tes t

3400 (750-18700)7000 (2100-44100)

p


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Carsin et al. : Evolut ion and signif icance of circulating procalcitonin levels 223

HO H4 H12 H I6 H20 H24 D2 D3* **

Time post burnFigure 8. Endotoxin concentrations (EU/ml) measured at each blood collection time. The area under the dotted line repre-sents the ran;ge of concentrations observed in the control group.

regardless of the cause. We also compared the IroCTpeak with the admission time IaOfiiOZ ratio as it isa marker of the pulmonary effector. We found nocorrelation between the l?aOfiiOZ ratio and theIroCT peak; in contrast, there was a clear correlationwith blood lactic acid levels.The origin of the early increase of ProCT remainsto be discovered. Our first hypothesis was that thisincrease was secondary to endotoxin secretion andgut bacter ial translocation. Injection of endotoxin tohealthy volunteers causes a rise in ProCT andtriggers an inflammatory reaction along withincreased circulating IL-6, TNFg and IL-1 levels3.Experimentally, in the burned animal there is arelationship between lowered mesenteric flowratedue to the burn and circulating endotoxin leve ls71E .Anumber of authorP found elevated circulatingendotoxin levels in men with severe burn injuries.Our previous studies have also shown the existenceof a correla tion between infection and high IroCTlevels at a later stage of the course of burn injuriesI.In 60 per cent of the patients of this study, we foundno circulating endotoxin during the early stage of theburn inju ry and no infection was observed in any ofthe 40 patients. Such results are in agreement withthose reported by Endo et a1.l. This does not rule ou tpossible bacterial translocation but, as proposed byMoore et al.lZ, one may th ink that in patientspresenting with polytrauma, the released bacteria areblocked in the mesenteric nodes where they arelike ly to induce macrophage activation. Hoch et al.13propose that the binding of circulating endotoxin tothe lipopolysaccharide binding proteins (LBP) maypreclude its detection in the plasma but does notprevent it from effecting its stimulating action. In

the critical discuss ion following Munsters studylo,Mannick emphasizes the difficulty in determining theendotoxin owing to contamination sources, plasmaactivators and inhibitors. For the purpose of ourobservations, all tubes were sterile and apyrogenic,the samples were treated under laminar air immedia-tely after collection and assays were validated bycalculating overload recoveries. Nevertheless, if therewas endotoxin production, one would expect to findhigh serum TNFu levels; we o,bserved this rise inonly one patient and he had no detectable plasmaticendotoxin. This absence of endotoxin in our patientsis perhaps explained by the fact that they all weretransported to the hospital in medically equippedvehicles where they received early vascular filling.Therefore, the early inflammatory reactionobserved in the patients with burn injuries seemsunrelated to gut bacterial translocation but simply toskin tissue alteration14J5. It is now well known thatkeratinocytes are initiators of inflammati0r-P. Theearly r ise in IroCT seems to be a marker of the massof destroyed tissue since the peak value observed inthe first 24 h is correlated with the UBS score.IL-6 levels provide reliable eviclence of this inflam-matory reaction9,17,1*. IL-6 elevation occurs as rapidlyas ProCT and they are statistically proven to bereliable markers of burn severityL*.ConclusionSerum procalcitonin and IL-6 levels rise quick lyfollowing severe burn inju ries. Peak levels observedin the first 24 h are related to burn severity but donot correlate with smoke inhalation injuries. System icincreases in procalcitonin do not seem to be related


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TV septic processes. We have been unable to find asignificant gut bacterial translocation or endotoxinrelease in the early hours following the thermalinjury. The origin of procalcitonin remains unknown.However, there is a clear relationship with thesystemic inflammatory response, the beginning ofwhich should be local, in relation to the mass ofdamaged tissues.

AcknowledgementsWe are grateful to Patrick Galaup, Fabrice Jaunault,,Ihilippe Verbeke and Olivier Gadal for their scientific co:ntribution; to Mr Arvers (Centre de Recherchesdu Service de Sante des Armees) for the statistical.analysis; and to Mrs Felten (Hopital Saint Louis) forher technical contribution.The present work was carried out thanks to aClin ical Research grant (RC 93/13) of the DirectionCentrake du Service de Sante des Armees.

References1 Assicot M , Gendrel D, Carsin H et al. High serum p rocalci-

tonin. concentrat ions in pat ients with sepsis and infect ion.Lancef 1993; 341: 515-518.

2 Davis THE , Assicot M, Bohuon C et al. Serum procalcitoninconcentrat ions in acute malaria. Trans R Sot Trap Med 1994;88: 670-671.3 Dandona I , Nix D, Wilson MF et al. Procalcitonin increasefol lowing endotoxin injection in normal subjects. I C&Endocrinol Metab 1994; 79: 1605.

4 ONeil l WJ , Jordan MH , Lewis MS et al. Serum calcitoninmay be a marker for inhalation injury in burns. J Burn CareRehabil 1992; 13: 605-616.

5 Skolnick A. Calcitonin assay may help ident ify burnpat ients at r isk for respiratory distress. JAMA 1990; 264:565-566.

6 Sachs A, Watson J. Four years experience at a special izedburn center. Lancef 1969; 1: 718.

7 Herndon DN , Zeugler ST. Bacterial t ransiocat ion afterthermal injury. Crif Cure Med 1993; 21: S50-S54.

8 Winchurch RA, Thupari JN, Munster AM. Endotoxin inburn pat ients. Surgery 1987; 102~ 808-812.9 Guo Y , Dickerson C, Chrest FJ et al. Increased level of

circulating interleukin 6 in burn patients. Clin lmrnunollmmunopafhol 1990; 54: 361-371.

10 Munster AM, S mith-Meek M, Dickinson C et al. Transloca-t ion, incidental phenomenon or true pathology?. Ann Surg1993; 218: 321-327.

11 Endo S, Inada K, Kikuchi M et al. Are plasma endotoxinlevels related to burn size and prognosis? Bt irrrs 1992; 18:486-489.

12 Moore FA, Moore EE, Pogett i R et al. Gut ba cterial t rans-location via the portal vein: a clinical persp ective withmajor trauma. J Trauma 1991; 31: 629-638.13 Hoch RC , Rodriguez R, Manning T et al. Effects ofaccidental t rauma on cytokin and endotoxin product ion.Crif Care Med 1993; 21: 839-845.

14 Cavail lon JM et al. Cytokines et inf lammation. In: Lescytokines. Paris: Masson S.A, 1993; p. 341.

15 Youn YK, Lalonde C, Demling R. Oxidants and the patho-physiology of burn and smoke inhalation injury. Free RadicBiol Me d 1992; 12~ 409-415.

16 Barker JN WN , M itra RS, Grif f i ths CEM et al. Kerat inocytesas init iators of inf lammation. Lancet 1991; 332 211-214.

17 Gueugniaud PY, Bert in-Maghit M, Joly MO et al. Role del interleukine 6 dans la phase oedemateuse du bnXe grave.Presse Med 1993; 22: 735.

18 Ueyama M, Maruyama I , Osame M et a l . Marked increasein plasma interleukin-6 in burn pat ients. J Lab Clip Med1992; 120: 693-698.

Paper accepted 26 September 1996.Correspondence should be addressed to: H. Carsin, Centre detraitement des brules, H.1.A Percy, BP 406, 92141 ClamartCedex, France.

46th Congresso Nazionale della SocietaItaliana di Chirurgia Plasti.ca Ricostruttivaed Estetica30 September-3 October 1997

Venice, IMyFor further information, contact:

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Carsin 1997 PCT Quemados