无标记红细胞形态学检测方法及其用于储存损伤的评估

无标记红细胞形态学检测方法及其用于储存损伤的评估无标记红细胞形态学检测方法及其用于储存损伤的评估

摘要：本研究旨在开发一种无标记的红细胞形态学检测方法，并通过该方法对红细胞贮存过程中可能导致储存损伤的因素进行评估。首先，通过比较已知浓度的标准红细胞与人体血液中的红细胞，确定了识别红细胞的亮度和颜色的临界值。然后，使用这些临界值对标准红细胞和血液中的红细胞进行检测，并将结果与现有方法进行比较。实验结果表明，无标记红细胞形态学检测方法具有更高的准确度和灵敏度，且不受特定标记物的限制。最后，使用该方法评估了红细胞贮存过程中低温、低氧、高盐度以及长期保存等因素引起的储存损伤。结果表明，储存时间越长，红细胞形态越不规则，表面微结构越粗糙，弹性降低，红细胞质膜蛋白分解程度增加等现象也逐渐出现，这些都是红细胞储存不可避免的损伤过程。

关键词：无标记、红细胞、形态学检测、储存损伤、评估

Introduction

Bloodtransfusionisanessentialmedicalpracticeworldwide,oftenusedintreatingacutebloodloss,severeanemia,andotherpathologicalconditions.Toensurethesafetyandefficacyofbloodtransfusion,donatedbloodistypicallystoredinspecializedbloodbanks.Duringstorage,redbloodcells(RBCs)undergovariousbiochemicalandbiomechanicalchangesthatcompromisetheirqualityandfunctionality.Thisprocessisknownasstoragelesionorstorage-relatedRBCinjury.StoragelesioncanleadtodecreasedRBCsurvival,impairedoxygen(O2)delivery,andincreasedriskofadversetransfusionreactions.Therefore,thedevelopmentofmethodstoassessstoragelesionandidentifyfactorsthatcontributetoitiscrucialforimprovingbloodtransfusionpractices.

Currently,severalmethodsareusedtoevaluatestoragelesion,includingmeasurementofhemolysis,pH,glucoselevels,andadenosinetriphosphate(ATP)degradation.However,thesemethodshavelimitations,suchaslowsensitivityandspecificity,interferencefromresidualanticoagulantsorpreservatives,andinabilitytodetectbiomechanicalchangesinRBCs.Therefore,thereisaneedformoreaccurateandcomprehensivemethodstoassessRBCstoragelesion.

RedbloodcellmorphologyisanessentialaspectofRBCfunctionandquality.RBCshapeandsizearecriticaldeterminantsoftheirdeformability,surfaceareatovolumeratio,andrheologicalproperties.AlterationsinRBCmorphologycanindicatevariouspathologicalconditions,includingRBCdisorders,infections,andautoimmunediseases.Moreover,RBCmorphologychangesduringstorage,reflectingtheeffectsofvariousstorageconditionsandthedurationofstorage.Therefore,RBCmorphologyisapotentialindicatorofstoragelesion.

ConventionalmethodstodetectRBCmorphologychangesrequirestainingorlabelingofRBCs,whichcanalterRBCproperties,resultincelldamageorloss,andinterferewiththeaccuracyofmeasurements.Therefore,thereisaneedfornon-invasiveornon-destructivemethodstodetectRBCmorphologychangesduringstorage.

Thisstudyaimedtodevelopanon-invasive,non-destructive,andlabel-freemethodtodetectandquantifyRBCmorphologychangesduringstorageandevaluatetheirrelationtostoragelesion.

Materialsandmethods

Bloodsampleswereobtainedfromhealthyvolunteerswhoprovidedwrittenconsent.Anticoagulant-treatedwholebloodsampleswerestoredinstandardbloodbankbagsat4ºCforupto42days.Atvarioustimepoints(days0,7,14,21,28,35,and42),sampleswerecollectedandprocessedforRBCmorphologyanalysis.

Acustom-madeimagingsystemwasusedtocapturebrightfieldimagesofRBCs.Imageswereanalyzedusingcustom-designedsoftwaretoextractinformationonRBCshape,size,andsurfaceroughness.ThesoftwareusedpredeterminedthresholdvaluesforbrightnessandcolortodifferentiateRBCsfromthebackgroundandotherbloodcomponents.

Results

TheproposedmethodwastestedonstandardRBCsuspensionsandwholebloodsamples.TheresultsshowedthatthemethodcouldaccuratelydetectRBCsanddistinguishthemfromotherbloodcomponents.Themethodwascomparedwithconventionalstainingmethodsandshowedsimilarresultsbutwaslesstime-consumingandlesspronetoerrorsduetostainingvariability.

TheRBCmorphologyanalysisresultsshowedthatRBCschangedtheirshape,size,andsurfaceroughnessduringstorage.Thechangesweremostevidentafter21daysofstorageandincreasedwithstorageduration.Thechangesincludedirregularshape,formationofspicules,lossofbiconcavity,andsurfacemembraneroughness.TheobservedchangescorrelatedwithdecreasedRBCdeformabilityandincreasedlysisandATPdegradation.

Conclusion

Theproposedlabel-freeRBCmorphologyanalysismethodisasensitive,accurate,andnon-invasivewaytoevaluateRBCmorphologychangesduringstorage.ApplyingthismethodtostoredbloodsamplesrevealedthatRBCundergoseveralalterationsthatareassociatedwithstoragelesion.TheresultssuggestthatstoragelesioncanbecharacterizedbychangesinRBCmorphology,whichcanbedetectedandquantifiedusingtheproposedmethod.Theproposedmethodhaspotentialapplicationsinassessingbloodstorageconditions,developingoptimalstoragestrategies,andimprovingtransfusionsafetyandefficacy.

Keywords:Label-free,redbloodcells,morphologyanalysis,storagelesion,assessmentRedbloodcells(RBCs)areacriticalcomponentofbloodtransfusion,andproperstorageofbloodisimportantforensuringtheirefficacyandsafety.However,duringstorage,RBCscanundergoseveralalterations,collectivelyknownasstoragelesion,whichaffectstheirfunctionalityandviability.ThesealterationsincludechangesinRBCmorphology,metabolism,membraneproperties,andhemoglobincontent.Amongthesealterations,changesinmorphologyareconsideredasoneofthemostsignificantandeasilydetectablechangesassociatedwithstoragelesion.

Toassesstheextentofstoragelesion,variousmethodshavebeendevelopedthatrelyondifferenttechniquessuchasflowcytometry,spectrophotometry,andmicroscopy.However,mostofthesemethodsrequirelabelingorstainingofRBCs,whichcaninterferewiththeirpropertiesandcompromisetheirfunctionality.Furthermore,labeling-dependentmethodsaretime-consumingandmayrequiresophisticatedequipmentandexpertise.Therefore,thereisaneedforalabel-freeandnon-invasivemethodthatcanaccuratelyassessRBCmorphologyanddetectstoragelesion.

Recently,alabel-freemethodhasbeenproposedforquantitativeanalysisofRBCmorphologybasedondigitalholographicmicroscopy(DHM).DHMisaninterferometrictechniquethatrecordstheinterferencepatternoflightwavesscatteredbyanobject,whichcanbeusedtoreconstructitsthree-dimensionalmorphologywithsubmicronresolution.TheproposedmethodutilizesDHMtocaptureholographicimagesofRBCsduringstorageandanalyzestheirmorphologyusingcomputervisiontechniques.TheresultsshowedthatDHM-basedmorphologicalanalysiscandetectandquantifychangesinRBCsize,shape,andtexture,whichareindicativeofstoragelesion.

Oneoftheadvantagesoftheproposedmethodisthatitislabel-freeanddoesnotrequireanysamplepreparationorstaining,whichminimizesthepotentialartifactsandvariabilityintroducedbylabeling-dependentmethods.Furthermore,themethodcanbeautomatedandhigh-throughput,allowingforrapidandaccurateassessmentofRBCmorphologyinlargequantitiesofsamples.Therefore,theproposedmethodhaspotentialapplicationsinassessingbloodstorageconditions,developingoptimalstoragestrategies,andimprovingtransfusionsafetyandefficacy.

Insummary,theproposedDHM-basedlabel-freemethodprovidesanon-invasiveandaccuratewaytoanalyzeRBCmorphologyanddetectstoragelesion.ThemethodhasthepotentialtoimprovebloodtransfusionsafetyandefficacybyenablingtheassessmentofbloodstorageconditionsandoptimizationofstoragestrategiesFurthermore,theDHM-basedlabel-freemethodhasthepotentialforwiderapplicationsinthefieldofbiomedicalresearch.Forexample,itcanbeusedtoanalyzethemorphologyofotherbloodcomponentssuchasplateletsandwhitebloodcells,whichcouldhelpinstudyingdiseasessuchassicklecellanemia,leukemia,andthrombocytopenia.Moreover,itcanbeutilizedtostudycelldynamicsandinteractionsinreal-time,whichcouldleadtoabetterunderstandingofcellbehaviorindifferentphysiologicalandpathologicalconditions.

AnotherpotentialapplicationoftheDHM-basedlabel-freemethodisinthefieldoftissueengineering.Tissueengineeringinvolvescreatingartificialtissuesthatcanbeusedforvariousmedicalpurposessuchasregenerativemedicineanddrugtesting.Themethodcanbeusedtostudythemorphologyandbehaviorofcellswithintheengineeredtissue,whichcouldhelpinoptimizingtissuefabricationandimprovingthefunctionalityofartificialtissues.

Inconclusion,theproposedDHM-basedlabel-freemethodisapromisingtoolforanalyzingRBCmorphologyanddetectingstoragelesion.Themethodhasthepotentialtoimprovebloodtransfusionsafetyandefficacy,andcouldhavewiderapplicationsinbiomedicalresearchandtissueengineering.Furtherstudiesarerequiredtovalidateitsefficacyindifferentclinicalandresearchsettings,andtooptimizeitstechnicalparametersforvariousapplicationsThisDHM-basedlabel-freemethodcanpotentiallyprovideadeeperunderstandingofRBCmorphologyandstoragelesionbyidentifyingsubtlechangesinthecells,suchasmembranedeformities,changesincellsizeandvolume,andhemoglobincontent.Furthermore,thenon-invasivenatureofthetechniqueeliminatestheneedforanysamplepreparation,whichreducestheriskofcontaminationandcanpotentiallypreservetheintegrityofthesamples.

ApartfromRBCanalysis,thismethodcanh