www.bjorl.org
Brazilian
Journal
of
OTORHINOLARYNGOLOGY
ORIGINAL
ARTICLE
Chemical
and
cytological
analysis
of
cerebral
spinal
fluid
after
intrathecal
injection
of
hypodense
fluorescein
夽
,
夽夽
Roberto
Eustáquio
Santos
Guimarães
a,∗,
Aldo
Eden
Cassol
Stamm
b,c,
Alexandre
Varella
Giannetti
d,
Paulo
Fernando
Tormin
Borges
Crosara
a,
Celso
Gonc
¸alves
Becker
a,
Helena
Maria
Gonc
¸alves
Becker
aaDepartmentofOtolaryngology,UniversidadeFederaldeMinasGerais(UFMG),BeloHorizonte,MG,Brazil
bDepartmentofOtorhinolaryngologyandHeadandNeckSurgery,UniversidadeFederaldeSãoPaulo(UNIFESP),SãoPaulo,SP,
Brazil
cDepartmentdeOtolaryngology,HospitalProfessorEdmundoVasconcelos,SãoPaulo,SP,Brazil
dDepartmentofNeurosurgery,UniversidadeFederaldeMinasGerais(UFMG),BeloHorizonte,MG,Brazil
Received29July2013;accepted13January2014
Availableonline21July2015
KEYWORDS
Fluorescein; Cerebrospinalfluid rhinorrhea; Leakage
Abstract
Introduction:Intrathecalfluoresceinhasbeeneffectivefortopographicdiagnosisof rhinoliqu-orrhea.Nonetheless,therearenoreportsonthestudyofcerebralspinalfluid(CSF)afteruse ofintrathecalfluorescein.
Objective: AprospectivestudyattemptingtoevaluateCSFthroughchemicalandcytological analysis,afterinjectionoffluorescein.
Methods:Prospective analysisof24samplesofCSF afterintrathecalinjectionoffluorescein fortopographicdiagnosisofCSFfistulae,collectedatthetimeofpunctureandafter24and 48h,dividedbycellularity:Group1,uptofivecells,andGroup2,withmorethanfivecells.
Results:Theyellow-greenishcolorofCSFremainedafter48hin36%,evidencingpermanence offluorescein.Nochangesinproteinandglucoselevelswereobserved between0---24hand 0---48h.Ingroup2,anincreaseincellcountwasobservedbetween24hand48h(p=0.019).In bothgroups,therewasanincreaseofneutrophilsbetween0and48h(p=0.048)andadecrease between24and48h(p=0.05).
夽
Pleasecitethisarticleas:GuimarãesRES,StammAEC,GiannettiAV,CrosaraPFTB,BeckerCG,BeckerHMG.Chemicalandcytological analysisofcerebralspinalfluidafterintrathecalinjectionofhypodensefluorescein.BrazJOtorhinolaryngol.2015;81:549---53.
夽夽
Institution:FaculdadedeMedicina,UniversidadeFederaldeMinasGerais(UFMG),BeloHorizonte,MinasGerais,MG,Brazil. ∗Correspondingauthor.
E-mail:[email protected](R.E.S.Guimarães). http://dx.doi.org/10.1016/j.bjorl.2015.07.016
Conclusion:Intrathecalfluoresceinprovokeddiscreetmeningealreactions,suchasanincrease ofcellsbetween24and48handanincreaseofneutrophilsat24h,withasubsequentdecrease at48hwithnocorrelationwithsymptomatology.
© 2015Associac¸ãoBrasileira de Otorrinolaringologiae CirurgiaCérvico-Facial. Publishedby ElsevierEditoraLtda.Allrightsreserved.
PALAVRAS-CHAVE
Fluoresceína; Líquido
cefalorraquidiano; Fístula
Análisequímicaecitológicadolíquorapósinjec¸ãointratecaldesoluc¸ãohipodensa defluoresceína
Resumo
Introduc¸ão:Afluoresceínaintratecaltemsidoefetivanodiagnósticotopográficoda rinoliquor-réia.Entretanto,nãoháestudosnolíquorapósousodefluoresceínaintratecal.
Objetivo:Estudoprospectivovisandoavaliarolíquor,atravésdeanálisequímicaecitológica, apósinjec¸ãodefluoresceína.
Método: Análiseprospectivade24punc¸õesapósinjec¸ãointratecaldefluoresceínapara diag-nósticotopográficodefístulaliquórica,coletadonomomentodapunc¸ão,24e48horas,divididos pelacelularidade:grupo1,comaté5célulasegrupo2commaisde5células.
Resultado:A colorac¸ão amarelo-esverdeada do líquor permaneceu após 48 horas em 36%, evidenciando permanênciade fluoresceína. Observou-se ausência de mudanc¸as no nível de proteínaeglicoseentre0---24horase0---48horas.Nogrupo2,umaumentonacontagemcelular foiobservadoentre24e48horas(p=0,019).Nodoisgruposjuntos,observou-seumaumento deneutrófilosentre0e48horas(p=0,048)eumadiminuic¸ãoentre24e28horas(p=0,05).
Conclusão:Fluoresceínaintratecal provocoudiscretas reac¸ões meníngeas, como oaumento decélulasentre24e48horaseaumentodosneutrófilosem24horascomumasubsequente diminuic¸ãoem48horassemcorrelac¸ãocomsintomas.
©2015Associac¸ãoBrasileira deOtorrinolaringologiaeCirurgiaCérvico-Facial.Publicadopor ElsevierEditoraLtda.Todososdireitosreservados.
Introduction
Cerebralspinal fluid(CSF)fistulae canoccurfromnose or externalearcanal,andfromatraumaticorsurgicaldefect inskullbase or spine. Fluidleakage is theresultof dural andarachnoidlaceration,withfistulaformation.CSF rhin-orrheaisclassicallydefinedasthepresenceofCSFinnasal
cavity, which implies the existence of a bone and dural
defectresultinginacommunicationbetweenthe
subarach-noidspaceandtheupperairwaycavities.1Thesesituations
requireaprecisediagnosisfortheiretiologyandlocation,2,3
togetherwithasuitabletreatment,duetoanincreasedrisk ofneurologicalcomplications,mainlybacterialmeningitis.
Althoughtherehavebeenseveraladvancesin preopera-tivetestsandinexaminationstodiagnose CSFrhinorrhea, theuseoffluorescein,whichbeganinthe1960s,hasbeen proveneffectiveastodetectionofsitesofCSFfistulae.4---9
However,reportsofadverse reactionsarefew,andare mainlyrelatedtohighconcentrations offluorescein;some centersarestillhesitanttousethisstrategyroutinely.10---18In
2001,afluoresceinsolutionwasdescribedinorderto facil-itateandhastentheidentificationoffistulasites.Previous studiesdescribingtheuseandsafetyofanintrathecal hypo-densefluoresceinsolutionhavealreadybeenpublished,but theyrarelyevaluatethechangesinCSFduetoitsuse.
Theobjectiveofthisstudywastodeterminethechanges inCSFafterusingahypodensefluoresceinsolution.Changes
incoloration,cellularity,andglucoseandproteinlevelsin CSFofpatientssubmittedtoitsusewereevaluated.
Methods
Thiswasaprospectivestudyof24consecutivepatientsaged 3---54 years (mean, 31 years) submitted to an intrathecal injection of hypodense fluorescein solution for an endo-scopic intraoperative diagnosis of CSF leakage from the anterioraspectofskullbase.
All patients provided informedconsent after a discus-sionoftherisks,benefits,andalternatives.Thestudywas approvedbytheEthicsCommitteeoftheinstitutionunder No.ETIC075/00.
Thirteenpatientsweremale(54%)and11female(46%). ThemaincauseofCSFrhinorrheawasatraumaticeventin 14 patients(58%), spontaneousrhinorrhea infive patients (21%), and meningocele or meningoencephalocele in five patients(21%).Theexclusioncriterionwastheoccurrence of meningitis in a previous period of three months. No patientswereexcludedfromthisstudy.
Allpatientsweresubmittedtoalumbarpunctureunder generalanesthesiaatthebeginningofsurgery.CSFwas col-lectedatthistimeforchemicalandcytologicalanalysis.
10mL of distilled water was injected. If the patient weighted less than50kg, or in children, 0.1mL/kgof the describedsolutionwasinjected.19 ThisstudyusedFluidag®
(sodium fluorescein, sodium bicarbonate, and phenylmer-curicnitrate;Oftalmopharma---SãoPaulo,Brazil).Fludiag®
consistsofasolutionof5%sodiumfluoresceindissolvedina physiologicallycompatiblebuffercontainingsodium bicar-bonateasalkalinizingagentinordertomaintainthepHin an8.0---9.8range,0.0001%phenylmercuricnitrateas preser-vative,andwaterforinjection.
Soon after the injection, which wasslowly performed over 2---3min, the patient was positioned supine, with his/herbackslightlyraisedatapproximately30◦;then,the
surgerywasinitiated.
After surgery and with the CSF fistula endoscopically closed,alumbardrainagecatheterinsertedfortheinjection offluoresceinwaskeptclosed.CSFsampleswerecollected at 24h and48h aftertheprocedure. These sampleswere submittedtochemical/cytologicalanalysis.
Chemical analysisof CSFincludedcolor (green, yellow-green, or clear), and glucose and protein levels by a colorimetricmethod.
Inthecytologicalanalysis,totalnumberoferythrocytes andnucleatedcells(neutrophils,lymphocytes,monocytes, eosinophils,andbasophils)wasobtained.ANeubauer cham-berwasusedforcytologicalanalysis.
Thepatientsweredividedintotwogroups,accordingto theirinitialnumberofcellsinCSF:Group1(uptofivecells permL)andGroup2(morethanfivecellspermL).These groupswerecompared,withtheaimtoexaminethe differ-encebetweenglucoseandproteinlevelsandcellularity.
Thestudydesignmadenoprovisionforacontrolgroup, thatis,onewithoutfluoresceininjection.
All results were statistically analyzed using Pearson’s coefficient,withstatisticalsignificancesetat95%(p<0.05).
Results
In all cases, hypodense fluorescein present in the endo-scopicsurgical fieldwasdetected, withsuccessfulclosure of CSFfistula. There wasnoneed to usespecial light fil-ters. There were no complications, adverse reactions, or meningitiscasesinthewholesampleof24patients.Group 1included14patientsandGroup2,tenpatients.
As toCSF staining, CSFsamples collected before fluo-rescein injection were clear in all patients. At 24h, in 20patients (80%) apredominantly yellow-greencolor was observed,indicating presenceoffluorescein.At48h, nine patients(36%) stillexhibitedayellow-greencolor in their CSFsamples.
Astoglucoselevel,therewasnostatisticallysignificant differencebetweenthetwogroupsaccessedconcomitantly atthezero-h(p=0.5),24-h(p=0.24),or48-h(p=0.5) mea-surements.
Regardingproteinlevel,therewasnostatistically signif-icantdifferencewithrespecttoproteinlevelsbetweenthe twogroupsanalyzedconcomitantlyatzero-h(p=0.37)and 24-h(p=0.16)measurements.Atthe48-hmeasurement,a statisticallysignificantdifferencewasobserved,with eleva-tionofproteinlevelsinGroup1(p=0.032).
120
100
80
Number of cells
Cells 0h
Cells 24h Cells 48h
Circles indicate outlier and asterisk (*) indicate an extreme value.
60
40
20
*
*
0
14 14
≤5
10 ≥6
14 10 10
Figure1 Cellularityinbothgroups(1and2)relatedtotime:
0h,24h,and48h.
10,0
8,0
6.0
Increase (percentage)
Increase 24h Increase 48h 4,0
2,0
0,0
–2,0
14
≤5 14 ≥6
Initial number of cells
10 10
Circles indicate outlier.
Figure2 Percentageofcellsinbothgroups(1and2)related
totime:0h,24hand48h.
Astocellcount,anincreaseinthisparameterwasalso observed.InGroup1,almostallpatientsshowedanincrease of600%,whencomparedtocellcountbaselinevalues.Half ofthe patientsshowed an increasein their cellcounts in excessof100%inbothgroups(Figs.1and2,Table1).
Among Group 2 patients, a statistically significant increaseincellcountwasobservedat48h.
Table1 Numberofcellsaccordingtotimecourseandinitialcellnumber.
Initialcellnumber Timeelapsedafterfluoresceininfusion p-value
0h 24h 48h 0hvs.24h 0hvs.48h 24hvs.48h
Mean SD Mean SD Mean SD
≤5 2.3 1.6 6.6 8.8 4.5 5.1 0.078 0.112 0.183
≥6 26.2 22.5 26.2 29.5 38.8 38.7 1.0000 0.213 0.019 Allpatients 12.3 18.6 14.8 22 18.8 30 0.493 0.117 0.117
p-values<0.05areinboldtype.
Table2 Percentageofneutrophilsaccordingtoprogressionoftimeandinitialcellnumber.
Initialcellnumber Timeelapsedafterfluoresceininfusion p-value
0h 24h 48h 0hvs.24h 0hvs.48h 24hvs.48h
Mean SD Mean SD Mean SD
≤5 13.2 29.9 41.7 35.9 24.2 26.6 0.118 0.459 0.108 ≥6 23.5 25.9 37.1 32.6 22.5 25.5 0.259 0.901 0.267 Allpatients 18.1 27.8 39.5 33.6 23.4 25.4 0.048 0.530 0.050
p-values<0.05areinboldtype.
100
80
Neutrophils at beggining
Neut 0h (%) Neut 24h (%)
Neut 48h (%) 60
40
20
0
12 12
None Present
12 9 9 9
Neutrophils at beggining
Figure3 Evolutionofpercentageofneutrophilsat0h,24h, and48hinrelationtoteststhatshowedneutrophilsat0hversus
thosewhodidnotpresentanyneutrophils.
Discussion
Intrathecal CSF fluorescein staining has been previously
described.6,7,12,15,16,19 This procedure allowsCSFdetection
frombloodorsecretionspresentintheoperatingfield.This justifiestheabsenceofacontrolgroup,i.e.without appli-cationoffluorescein.
Intrathecalapplicationoffluoresceinisan off-labeluse of this product and its use requires documentation and a specific discussion with the patient,requiring informed
written consent.8 Severaladverse eventssuchasseizures,
transientparalysis,andneuropathicpainhaveoccasionally beenattributedtoitsuseinliterature.11,12,15Theincidence
ofcomplications remainsextremelylow andsomeauthors reportedhigherpercentagesinpatientswhoreceivedhigher or more concentrated doses than that described in this study.15---19 The pathophysiology of these events remains
unclear,buttheymaybesecondarytoameningeal inflam-mation,whichchangesthechemistryofnormalCSFandits cytology.8,12,15
NormalCSFhasaproteincontentofabout20---45mg/dL. Theglucoselevelinthismediumisabout50---100mg/dL;CSF maycontainuptofivecellspermL.Thesewerethevalues usedinthisstudy,inordertoperformthisevaluation.20
No adverse reactions were observed with the use of an intrathecal hypodense fluorescein solution at the dosage describedin thisstudy,evenin children.Withthis dosage,theskullbasedefect visualizationwasconsidered effective.16,19
Nevertheless, there were chemical and cytological changesinCSF.In36%ofpatients,CSFcolor,whichwasclear inthefirstcollection,changedwiththeuseoffluorescein, up to48h afterinjection. This observation can be corre-latedwiththefindingsofcellprogressionandpercentageof neutrophils.
ThepresenceoffluoresceininCSFcanactasatriggering factorforneutrophiliaandincreasedcellcount,aswellas totheirsubsequentdecreaseafterthefluoresceinlevelsin CSFdiminished.
phenylmercuricnitrateandsodiumbicarbonatemaycause someofthechangesobservedinthisstudycannotberuled out. Nonetheless, the cytological changes observed were minimal,andwerenotassociatedwithsideeffects.Besides, thesechangescanbeevensmaller,dependingonthe pres-enceofthesecompoundsinthesolutionused.
TherewerenosignificantchangesinglucoselevelsinCSF. Thisfindingisimportant,sincenoneofthepatientsshowed anysymptomofbacterialmeningitisduringthestudy.Onthe otherhand,proteinlevelsfromGroup1patientsincreased atthe24-hand48-h(p=0.032)measurementsvs.Group2 patients,whichcanbeexplainedbytheincreasednumber ofcellspresentintheCSFsamplesofGroup1.
Conclusion
Based on these results, it can be concluded that the intrathecal hypodense fluorescein solution(Fluidag®)
pro-motes chemical and cytological changes in CSF, with an increaseincellnumber(mainlyat24hand48h)andin neu-trophilsat 24hwith subsequentdecreaseat 48h.Protein levelsincreasedat24hand48h.Therewerenochangesin glucoselevels.Nevertheless,thesechangesdidnottranslate intoanyclinicalmanifestations.
Conflicts
of
interests
Theauthorsdeclarenoconflictsofinterest.
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