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THEIMPORTANCETHEIMPORTANCEOFAGOODH&E-STAINEDSLIDE:JohnK.C.QueenElizabethHospitalHongKong,SARChinaInsurgicalInsurgicalpathologyagoodH&EsectionisessentialforaproperdiagnosisThisisthemoreimportantforlymphoidlesions--goodcytologicdetailsareimportantfordiagnosisorclassificationEventopexpertcannothelpifslidesareGoodGoodqualitysectioningandGoodGoodqualitysectioningandGoodGoodqualityGoodGoodqualityH&E:3tonesofredNotebrillianceofcolor,sharpcontrastandexquisiteThreetonesThreetonesofredNoteNoteabsenceofemptyspacesaroundindividual NoteNoteabsenceofemptyspacesaroundindividualNucleishowgoodchromatinNoteNotechromatindetailsandnuclear-cytoplasmicNoteNotechromatindetailsandnucleus-cytoplasmLowLowtomedium-power:sectionsofusualthickness(4µm)toassessarchitectureHighHighthinsectionforcytologicMF:MF:Thinsectionsrevealnucleardetailsmuch LowLowtomedium-power:sectionsofusualthickness(4µm)toassessarchitectureHighHighthinsectionforcytologicAmphophilicAmphophilic/basophiliccytoplasm:EosinophilicEosinophiliccytoplasm:Histiocytic/MajorfactorsMajorfactorsthatmayaffectqualityofH&E-stainedslides(I)•TissuefixationandBufferedformalin(acidicformalinleadstodepositionofformalinpigment)Promptfixation(cutopenlargerAdequatetissuedehydrationduringtissueprocessing(smallspecimensrequireshorterdehydrationsteps,otherwisecellsmayappearshrunken)FormalinFormalinProminentProminentshrinkageartifactsfromdelayedPoorPoorfixation/processing,withshrinkage DelayedDelayedfixation,withshrinkage PoortissuePoortissuefixationandpoorLN:LN:GoodH&Eforcomparison(noretraction AllAllcellssurroundedbyretractionMajorfactorsMajorfactorsthatmayaffectqualityofH&E-stainedslides(II)•TissueAdequatehydrationofparaffinblocksbeforeAdequateexpansionofparaffinsectioninwaterGoodmicrotomytoobtainasectionofuniformthickness,freeofscratchesNPNPbiopsy:InadequatehydrationofparaffinNPNPbiopsy:IncreasedhydrationofparaffinInadequatehydrationInadequatehydrationofIncreasedhydrationofInadequateInadequatehydrationofIncreasedhydrationofPoorPoorThickThicksection:difficulttoappreciatecytologicThickThicksection:difficulttoappreciatecytologicMajorMajorfactorsthatmayaffectqualityofH&E-stainedslides(III)H&EAdequatehematoxylinAcid-alcoholdifferentiationstepveryimportant(nottooconcentrated,andnottoodilute;weuse0.3%)Adequateeosinstaining;dilutedalcohol(fromcarry-overofwater)inthesubsequentstepsmaydecolorizetheredcolorTypeofhematoxylindetermineswhetheracidicmucinisstainedup(Mayerhematoxylindoesnotstainacidicmucin–mayloseimportantdiagnosticinformation)•PoorPoorcontrastandcytologicPoorPoorcontrastandcytologicPoorPoorcontrastandcytologicWouldWouldhavebeenbetteriftheeosinisTooToomucheosin;insufficient TooToomucheosin;insufficient DigitalDigitalMayerBreastBreastmucinousMayerMajorMajorfactorsthatmayaffectqualityofH&E-stainedslides(IV)Dehydrationand–Dryingofslideafterstaining(e.g.usingheattodryslideinplaceofxylene)resultsinsignificantartifacts•AAbadH&E-stainedslidecannotbecompensatedbyothertechniques,suchasimmunohistochemistryormolecularstudiesForaForalongtime,wehavenoticedsomepoorH&E-stainedslidesfromChina(includingfromsomemajorcenters)SuchartifactshavenotbeenencounteredinH&EslidessentinfromothercountriesoftheworldProblemmustbeduetosomefactor(s)uniquetoChina,e.g.reagents,localpracticeSwollenSwollenandsmudgynuclei;poorShrunkenShrunken ShrunkenShrunken OriginalOriginalSectionstainedinOriginalOriginalSectionstainedinOriginalOriginalSectionstainedinOriginalOriginalSectionstainedin Problemisnotrelatedtotissuefixation,tissueprocessingProblemisnotrelatedtotissuefixation,tissueprocessingorslidecuttingprocedureSoproblemliesinoneormorepost-sectioning網網上討論網上討論網上討論DryingbyDryingbyhair-dryer!RoutineDryingbyDryingbyhair-dryer!RoutineRoutineDryingRoutineDryingbyhair-dryer!directDryingbyDryingbyhair-RoutineRoutineDryingRoutineDryingbyhair-afterafterH&EstainingresultsinpoorandunacceptablemorphologySuchartifactsaresimilarirrespectiveofthemethodortemperatureofdrying(e.g.hair-dryer,oven,heatingchamber)•DepartmentsDepartmentsknowntoustohaveproblematicH&Estains!H&EH&Eslidesof2hospitalsaftermodificationsofH&Estainingprotocol(nomoredryingofslides)SlideSlidedryingafterstainingnotonlyimpactsH&E,butalsoadverselyimpactsimmunostainedslides!Drying SuboptimalSuboptimalH&E-stainedslidesadverselyaff