I-new methylene blue nokudaya ii-reticulocyte: idayi eyahlukileyo nendlela yeeseli eziphilayo

Umfundi ulungiselela ukubala ii-reticulocyte, aodole i-methylene blue, ayixube negazi, aze angaboni phantse nto. Omnye umfundi usebenzisa ibhotile ebhalwe i-new methylene blue aze abone imicu eluhlaza okwesibhakabhaka ecacileyo ngaphakathi kwezinye iiseli ezibomvu zegazi. Umfundi wokuqala ucinga ukuba idayi ekwibhotile yesibini intsha ngakumbi okanye inoxinzelelo oluphezulu. Akunjalo. Iqulethe imolekyuli eyahlukileyo.

Le mpazamo yokuqonda ingunobangela woninzi lokubhideka kulo mbandela. Igama libonisa ngathi i-new methylene blue yibhetshi ephuculweyo ye-methylene blue. Kodwa ikhemistri ibonisa okwahlukileyo. I-new methylene blue N yidayi ye-phenothiazine eyahlukileyo, enenombolo yayo kwi-Colour Index, ilungiselelo layo lamaqela abambe indawo yeeathom, kunye nendima yayo esemgangathweni kufundo lwegazi. Xa lo mahluko ucacile, yonke enye inkqubo — isizathu sokuba iiseli zimele ziphile xa zidaywa, into ebalwa njenge-reticulocyte, indima yemizimba ye-Heinz, nexesha esetyenziswa ngalo i-brilliant cresyl blue — iba lula ukuyiqonda.

Impendulo emfutshane

I-new methylene blue N, isixhobo sekhemikhali esisetyenziswa kufundo lwegazi, idla ngokuchongwa njenge-Basic Blue 24 kwirekhodi ye-PubChem ye-new methylene blue, C.I. 52030, CAS 1934-16-3, enefomula ethi C18H22ClN3S. I-methylene blue eqhelekileyo yi-methylthioninium chloride, i-Basic Blue 9, kwirekhodi yayo ye-PubChem, C.I. 52015, CAS 61-73-4, enefomula ethi C16H18ClN3S. Ngokwesakhiwo, i-new methylene blue inamaqela amabini e-methyl akwisangqa semolekyuli kunye namatyathanga asecaleni e-ethylamino kwiindawo apho i-methylene blue inamaqela e-dimethylamino. Lo mahluko utshintsha indlela edaya ngayo. Iincwadi zokufunda ngegazi zichaza i-methylene blue eqhelekileyo njengedayi ebuthathaka yokudaya ii-reticulocyte, zize zichaze i-new methylene blue njengedaya ngamandla nangokulinganayo ngakumbi; olu thelekiso lushwankathelwe kumxholo we-ScienceDirect nge-new methylene blue.

Ewe, i-new methylene blue yidayi esetyenziswa kwiiseli ezisaphilayo emva kokuba zikhutshwe emzimbeni, oko kubizwa ngokuba yi-supravital staining. Ukudaya ngale ndlela kuthetha ukudaya iiseli eziphilayo emva kokuba zikhutshwe emzimbeni nangaphambi kokuba zilungiswe ukuze zingaguquki, njengoko kuchazwe kuphononongo lwe-NCBI lwemigaqo yokudaya nge-supravital. Zombini i-new methylene blue ne-brilliant cresyl blue zikolu didi, yaye umgangatho we-CLSI wee-reticulocyte ubhekisa ngokucacileyo kwi-new methylene blue njengedayi ye-supravital. Ngamanye amaxesha abafundi bayidibanisa nokudaya nge-Romanowsky kwizithambiso zegazi esele zilungisiwe ukuze zingaguquki. Loo ndlela yokucinga ayisebenzi apha. Ukuba uqala ngokuzilungisa ukuze zingaguquki, le ndlela ayisasebenzi.

Kubafundi abafuna ukwahlula amagama eedayi asondeleleneyo, inqaku elithi i-methyl blue xa ithelekiswa ne-methylene blue lichaza ukubhideka okwahlukileyo nokukhulu ngakumbi phakathi kwedayi ye-triarylmethane enentlawulo yombane engathabathiyo nosapho lwe-phenothiazine olunentlawulo yombane evumayo. Isishwankathelo sokudaya elabhoratri sibeka umsebenzi we-supravital phakathi kweendlela ezisebenzisa izithambiso esele zilungisiwe ukuze zingaguquki, kanti isikhokelo seemilo zeeseli zegazi sibonisa indawo yovavanyo lwee-reticulocyte ekuhlukaniseni ngokubanzi iintlobo zeeseli zegazi.

Isilumkiso esinye ngeekhathalogu: kukho uhlobo olwahlukileyo lwe-new methylene blue oluyityuwa ephindwe kabini ye-zinc chloride phantsi kwe-CAS 6586-05-6. Ngoko icandelo leendlela kufuneka lichaze uhlobo lwetyuwa nomthengisi, kungabi ligama eliqhelekileyo kuphela.

Okwenziwa ngokwenene kukudaya nge-supravital

I-reticulocyte yiseli encinane ebomvu yegazi esele ilahlekelwe yinucleus yayo kodwa isenentsalela ye-RNA yeeribosome. Kwisampulu emanzi engadaywanga, loo RNA ayibonakali. Idayi ye-supravital ingena kwiseli ephilayo ize ikhuphe loo RNA kwisisombululo okanye iyidibanise ibe ngamasuntswana nemicu eluhlaza okwesibhakabhaka, nto leyo eyenza izinto ezinjengomnatha nemicu ezibonwa kusetyenziswa ioyile phakathi kwesampulu nelensi. Ngoko loo mnatha uveliswa kukudaya; awusiso isakhelo esele sikhona esilindele ukufakwa umbala. Yiyo loo nto izinto eziguquguqukayo kwinkqubo — uxinzelelo lwedayi, isithinteli sokutshintsha kwe-pH, umlinganiselo wegazi nedayi, iqondo lobushushu nexesha — zibaluleke ngokufanayo negama ledayi.

Oku kuthetha ukuba amanyathelo kufuneka alandele ulandelelwano olungqongqo: qala uxube idayi negazi elifakwe into ethintela ukujiya, uliyeke lisebenze kangangexesha elimiselweyo, emva koko wenze izithambiso uze uzihlole zingalungiswanga ukuze zingaguquki kwaye zingafakwanga idayi yesibini. Ukuzilungisa ukuze zingaguquki ngaphambi kokudaya, okanye ukufaka idayi yesibini esitha imicu enombala obuthathaka, kutshabalalisa ulwazi oluxhomekeke kulo ubalo.

Inkqubo esebenzayo yee-reticulocyte nge-new methylene blue

Akukho mxube mnye osetyenziswa kuyo yonke indawo, ngoko chaza lowo usetyenzisiweyo. Iindlela ezintathu ezibhaliweyo zibonisa umahluko okhoyo.

Inkqubo yakudala yohlobo lwe-Brecher isebenzisa malunga ne-0.5 ekhulwini ye-new methylene blue ene-potassium oxalate, ixutywe ngomthamo olinganayo negazi le-EDTA elixutywe kakuhle, ize iyekwe isebenze malunga nemizuzu eli-10 ukuya kweli-15 kubushushu begumbi. Emva kokuyixuba kwakhona ngobunono, kwenziwa izithambiso ezibhityileyo ezimile njengeweji, zomiswe ngomoya, zize zihlolwe zingalungiswanga ukuze zingaguquki kusetyenziswa ilensi ye-100x eneoyile. Inkqubo ye-NCBI yokubala ii-reticulocyte ichaza la manyathelo kunye nesakhelo sokubala esisetyenziswa nawo.

Indlela ye-Dacie ne-Lewis isebenzisa i-1.0 ekhulwini ye-new methylene blue kwisisombululo setyuwa esine-citrate, ixutywe negazi elinomthamo ophindwe ka-2 ukuya ka-4, ize igcinwe ku-37 C kangangemizuzu eli-15 ukuya kwengama-20 ngaphambi kokwenza izithambiso. Ukuphunyezwa kwakutshanje kokulungelelanisa inkqubo ye-CLSI H44 kusebenzise isisombululo esinoxinzelelo olusezantsi lwe-0.1 ekhulwini kwisisombululo setyuwa esine-phosphate esithintela ukutshintsha kwe-pH, kwi-pH 7.4, sayekwa sisebenze ku-20 C kangangemizuzu eli-10, ngaphandle kokulungiswa ukuze singaguquki, njengoko kuchaziwe kuphononongo luka-2026 lokulungelelanisa i-NMB.

Imithetho yokubala iyafana kuzo zonke ezi ndlela. Bala iseli njenge-reticulocyte xa inamasuntswana adayiwe aluhlaza okwesibhakabhaka ubuncinane amabini ahlukeneyo okanye iziqwenga zemicu, nto leyo engabandakanyi idiphozithi yedayi eyodwa. Bala ubuncinane iiseli ezibomvu zegazi eziyi-1,000 kumsebenzi wesiqhelo, uze uchaze ii-reticulocyte njengepesenti. Xa iipesenti ziphantsi, iinkcukacha-manani zifuna kubalwe iiseli ezininzi ngakumbi. Uphononongo lwendlela ye-CLSI H44 luphawula ukuba xa ii-reticulocyte zimalunga ne-1 ekhulwini, kufuneka malunga neeseli eziyi-4,400 ukuze umahluko kubalo wehle uye kwi-5 ekhulwini, kuphononongo lwe-CLSI H44 oluse-De Gruyter. Idiski ye-Miller ekwilensi yokujonga yenza kube lula ukwenza ubalo olukhulu. Hluza idayi okanye uyilungise isentsha, kuba idayi eqokelelene ngaphandle kwesisombululo ifana namasuntswana angaphakathi kwiiseli.

Imizimba ye-Heinz kwisampulu enye

Kwaeso sithambiso se-supravital sinokubonisa nemizimba ye-Heinz, eziziqokelelo zehemoglobin eyonakaliswe yioksideshini yaza yatshintsha isakhiwo sayo, kungekhona i-RNA eshiyekileyo. Kwiisampulu ezilungiselelwe nge-new methylene blue ibonakala njengezakhiwo ezingqukuva, ezidla ngokuba nye kwaye zibe kufutshane nenwebu yeseli, zidaywe ngombala oluhlaza okwesibhakabhaka okhanyayo ngakumbi kunowezinto ezinjengomnatha nemicu. Ayigcineki kakuhle xa isithambiso silungiswa ukuze singaguquki naxa kufakwa idayi yesibini, ngoko hlola isithambiso singalungiswanga ngolo hlobo. Inkqubo yakutshanje esebenzisa iimicroliter ezingama-20 zegazi le-EDTA kunye neemicroliter ezingama-20 ze-new methylene blue ye-1 ekhulwini, zize ziyekwe zisebenze kangangemizuzu eli-15, ibale iiseli ezinemizimba ye-Heinz phakathi kwee-erythrocyte ezingama-250, kuphononongo lwe-PMC oluchaza loo nkqubo.

Umahluko ombini uthintela ukutolika ngendlela engachananga. Okokuqala, i-methyl violet ne-crystal violet zidaya imizimba ye-Heinz ngamandla ngakumbi kune-new methylene blue, njengoko kuphawuliwe kumxholo we-ScienceDirect ngemizimba ye-Heinz. I-new methylene blue lukhetho olulungele ukusetyenziswa xa ii-reticulocyte nemizimba ye-Heinz zivavanywa kunye; ayisiyodayi enamandla kakhulu eyenzelwe ngokukodwa imizimba ye-Heinz. Okwesibini, izinto ze-HbH ezingaphakathi kwiiseli zabantu abanophawu lwe-alpha thalassemia zivelisa amachaphaza amaninzi amancinane kuyo yonke iseli emva kokudaya nge-supravital, ngoxa imizimba ye-Heinz eqhelekileyo imbalwa, mikhulu ngakumbi, kwaye idla ngokunxulumana nenwebu yeseli. Idiphozithi yedayi yinto yesithathu efana nayo: iba ngaphandle komaleko ekugxilwe kuwo weeseli kwaye iyahluka phakathi kweendawo ezijongwayo.

I-brilliant cresyl blue xa ithelekiswa ne-new methylene blue

Zombini ezi dayi zisetyenziselwa ukudaya ii-reticulocyte zisaphila, kodwa ukumiselwa kwemigangatho kukhetha enye yazo. Indlela yee-reticulocyte ye-CLSI H44-A2, eyaphuhliswa ngokubambisana ne-International Council for Standardization in Haematology neyidweliswe kwiphepha lomgangatho we-CLSI H44, ichaza ukudaya igazi le-EDTA nge-new methylene blue njengendlela yokuthelekisa eyenziwa ngesandla. Ukufundiswa kwakudala kufundo lwegazi kuyavumelana ngenkangeleko: i-new methylene blue idaya imicu ngamandla nangokulinganayo ngakumbi, ngoxa i-brilliant cresyl blue ihluka ngakumbi phakathi kweebhetshi neesampulu.

Elo lungelo layo kufuneka licaciswe ngemida yalo. Isikhokelo se-ICSH sokuvavanya izixhobo zokuhlalutya samkela uhlolo lwemikroskopu olwenziwa ngesandla nge-new methylene blue njengendlela yokuthelekisa nesalathiso, kodwa sithi luxhomekeke ekutolikeni komntu kwaye aluchanekanga ngokwaneleyo xa luthelekiswa nezinye iindlela; sikhetha iinkqubo zesalathiso ze-flow cytometry apho zifumaneka khona. Uthelekiso luka-2022 lweesampulu ezili-100 lufumene ukuba i-brilliant cresyl blue inomahluko ocwangcisiweyo omncinane kwiziphumo zesixhobo sokuhlalutya esisetyenzisiweyo kunowe-new methylene blue, njengoko kuchaziwe kuthelekiso lwe-PMC lweendlela zokudaya. Isishwankathelo esinokuthethelelwa sabafundi sesi: sebenzisa i-new methylene blue njengendlela esemgangathweni yokuthelekisa eyenziwa ngesandla, qinisekisa nayiphi na indlela ye-brilliant cresyl blue esetyenziswa kwindawo yakho ngokuyithelekisa nayo okanye nenkqubo ezenzekelayo, uze ulindele ukusasazeka okubanzi ngakumbi kweziphumo zohlolo lwemikroskopu olwenziwa ngesandla, nokuba yeyiphi kwezi dayi esetyenzisiweyo. Uncwadi oludala lokumisela imigangatho lwalukwakhuthaza i-Azure B ecociweyo njengenye indlela ecoceke ngakumbi ngokwekhemikhali, kuba iibhetshi zeedayi ezithengiswayo zazihluka, njengoko kuchaziwe kuphononongo luka-Marshall nge-Azure B, nto leyo echaza isizathu sokuba inqanaba lococeko lwesixhobo sekhemikhali lisaxoxwa kwiindlela zokusebenza.

Ukuphepha iimpazamo eziqhelekileyo

Iimpazamo ezine zingunobangela woninzi lweesampulu ezingalungiselelwanga ngempumelelo. Ukuodola i-methylene blue eqhelekileyo endaweni ye-new methylene blue N kubangela ukudaya okubuthathaka ngenxa yekhemistri, kungekhona ngenxa yendlela yokusebenza. Ukulungisa isithambiso ukuze singaguquki okanye ukusifaka idayi yesibini ngaphambi kokusihlola kususa umqondiso we-supravital. Ukubala idayi eqokelelene ngaphandle kwesisombululo njengamasuntswana kunyusa isiphumo ngokungeyonyani; umthetho wamasuntswana amabini kunye nedayi ehluziweyo kuthintela oku. Ukubala iiseli ezimbalwa kakhulu xa iipesenti zee-reticulocyte ziphantsi kuvelisa inani elibonakala lichanekile kodwa elinemida ebanzi yokuqiniseka ngokweenkcukacha-manani. Impazamo nganye inophawu olufanayo: umntu ohlola ngemikroskopu ubeka ityala kwim ikroskopu ngenxa yento esele igqitywe linyathelo lesixhobo sekhemikhali.