無標記紅細胞形態(tài)學檢測方法及其用于儲存損傷的評估

無標記紅細胞形態(tài)學檢測方法及其用于儲存損傷的評估無標記紅細胞形態(tài)學檢測方法及其用于儲存損傷的評估

摘要：本研究旨在開發(fā)一種無標記的紅細胞形態(tài)學檢測方法，并通過該方法對紅細胞貯存過程中可能導致儲存損傷的因素進行評估。首先，通過比較已知濃度的標準紅細胞與人體血液中的紅細胞，確定了識別紅細胞的亮度和顏色的臨界值。然后，使用這些臨界值對標準紅細胞和血液中的紅細胞進行檢測，并將結果與現有方法進行比較。實驗結果表明，無標記紅細胞形態(tài)學檢測方法具有更高的準確度和靈敏度，且不受特定標記物的限制。最后，使用該方法評估了紅細胞貯存過程中低溫、低氧、高鹽度以及長期保存等因素引起的儲存損傷。結果表明，儲存時間越長，紅細胞形態(tài)越不規(guī)則，表面微結構越粗糙，彈性降低，紅細胞質膜蛋白分解程度增加等現象也逐漸出現，這些都是紅細胞儲存不可避免的損傷過程。

關鍵詞：無標記、紅細胞、形態(tài)學檢測、儲存損傷、評估

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