Isihloko
I-new methylene blue nokudaya ama-reticulocyte: udayi ohlukile nendlela yamaseli aphilayo
Umfundi ulungiselela ukubala ama-reticulocyte, a-ode i-methylene blue, ayixube negazi, bese ebona cishe kungekho lutho. Omunye umfundi usebenzisa ibhodlela elibhalwe ukuthi new methylene blue bese ebona imicu eluhlaza okwesibhakabhaka ecacile ngaphakathi kwamanye amaseli abomvu egazi. Umfundi wokuqala ucabanga ukuthi udayi osebhodleleni lesibili usanda kwenziwa noma ugxile kakhulu. Akunjalo kokubili. Uqukethe i-molecule ehlukile.
Leli yiphutha lokuqonda elibangela ukudideka okuningi kule ndaba. Igama lisikisela ukuthi i-new methylene blue iyiqoqo elithuthukisiwe le-methylene blue. Ikhemistri ikhombisa okuhlukile. I-new methylene blue N ingudayi we-phenothiazine ohlukile, onenombolo yawo ye-Colour Index, ukuhleleka kwawo kwamaqembu athatha indawo esakhiweni samakhemikhali, nendima yawo ebekwe ngokwezinga elifanayo ocwaningweni lwegazi. Uma lowo mehluko usucacile, konke okunye ngendlela yokusebenza kuba lula ukukuqonda: kungani amaseli kufanele aphile lapho edaywa, yini ebalwa njenge-reticulocyte, imizimba ka-Heinz ingena kuphi, nokuthi i-brilliant cresyl blue isetshenziswa nini.
Impendulo emfushane
I-new methylene blue N, i-reagent esetshenziswa ocwaningweni lwegazi, ngokuvamile ihlonzwa ngokuthi Basic Blue 24 kurekhodi le-PubChem le-new methylene blue, C.I. 52030, CAS 1934-16-3, ifomula C18H22ClN3S. I-methylene blue evamile iyi-methylthioninium chloride, Basic Blue 9, ngokwerekhodi layo le-PubChem, C.I. 52015, CAS 61-73-4, ifomula C16H18ClN3S. Ngokwesakhiwo, i-new methylene blue inamaqembu amabili e-methyl eringini namaketanga aseceleni e-ethylamino lapho i-methylene blue inamaqembu e-dimethylamino. Lowo mehluko ushintsha indlela udayi osebenza ngayo. Izincwadi zocwaningo lwegazi zichaza i-methylene blue evamile njengodayi ongadayeli kahle kakhulu ama-reticulocyte, kanti i-new methylene blue idaya ngokujulile nangokulinganayo kakhulu; lokhu kuqhathaniswa kufingqwe esihlokweni se-ScienceDirect nge-new methylene blue.
Yebo, i-new methylene blue ingudayi we-supravital. Ukudaya nge-supravital kusho ukudaya amaseli aphilayo ngemva kokuwakhipha emzimbeni nangaphambi kokuwafiksa, njengoba kuchazwe ekubuyekezweni kwe-NCBI kwezimiso zokudaya nge-supravital. Kokubili i-new methylene blue ne-brilliant cresyl blue kungena kulesi sigaba, futhi izinga le-CLSI lama-reticulocyte likhuluma ngokuqondile nge-new methylene blue njengodayi we-supravital. Ngezinye izikhathi abafundi bakuhlanganisa nokudaya nge-Romanowsky amasampula asatshalaliswe aba izingqimba ezincane futhi afiksiwe. Leyo ndlela yokucabanga ayisebenzi lapha. Uma ufiksa kuqala, le ndlela iyeka ukusebenza.
Kubafundi abalandela amagama odayi asondelene, ukuqhathaniswa kwe-methyl blue ne-methylene blue kuchaza ukudideka okuhlukile nokukhulu phakathi kukadayi we-triarylmethane one-charge engemihle nomndeni we-phenothiazine one-charge enhle. Uhlolojikelele lokudaya elabhorethri lubeka umsebenzi we-supravital phakathi kwezindlela ezisebenzisa izingqimba zamaseli ezifiksiwe, kanti umhlahlandlela wokwakheka kwamaseli egazi ukhombisa ukuthi ukuhlolwa kwama-reticulocyte kungena kuphi ekuhlukaniseni izinhlobo zamaseli egazi okubanzi.
| I-reagent | Ubunjalo | C.I. / CAS | Indima emsebenzini wama-reticulocyte |
|---|---|---|---|
| New methylene blue N | I-phenothiazine enamaqembu e-ethylamino namaqembu amabili e-methyl eringini | C.I. 52030 (Basic Blue 24); CAS 1934-16-3 ye-chloride | Udayi we-supravital obekwe ngokwezinga elifanayo wokudaya ngesandla, okhethwayo njengesisekelo sokuqhathanisa |
| I-methylene blue evamile | I-bis(dimethylamino) phenothiazine (methylthioninium chloride) | C.I. 52015 (Basic Blue 9); CAS 61-73-4 | Udayi ongadayeli kahle ama-reticulocyte, ongeke usetshenziswe esikhundleni sawo |
| Brilliant cresyl blue | Udayi we-oxazine, ukwakheka kwawo kuyahluka ngokomphakeli | C.I. 51010; CAS 81029-05-2 noma 10127-36-3 kuye ngohlobo lukasawoti | Udayi we-supravital owamukelekayo, ongadayeli ngokulinganayo kangako, ongeyona indlela yokuqhathanisa evunyelaniswe ngokwezinga |
Isixwayiso esisodwa ngamakhathalogi: kukhona uhlobo oluhlukile lwe-new methylene blue olungusawoti ophindwe kabili we-zinc chloride ngaphansi kwe-CAS 6586-05-6. Ngakho-ke isigaba esichaza izindlela kufanele sisho uhlobo lukasawoti nomphakeli, hhayi igama elijwayelekile kuphela.
Lokho ukudaya nge-supravital okukwenzayo ngempela
I-reticulocyte iyiseli elibomvu legazi elisencane elilahlekelwe yinucleus yalo kodwa elisenayo i-RNA eyinsalela yama-ribosome. Esampuleni emanzi engadaywanga, leyo RNA ayibonakali. Udayi we-supravital ungena eselini eliphilayo bese wenza leyo RNA iphuma esixazululweni noma ihlangane ibe yizinhlayiyana nemicu eluhlaza okwesibhakabhaka, okuwukwakheka kwenethiwekhi nemicu okubonwa ngokusebenzisa uwoyela ngaphansi kwesibonakhulu. Ngakho-ke leyo nethiwekhi ingumkhiqizo wokudaya, hhayi uhlaka olwakhiwe kusengaphambili olulindele ukufakwa umbala. Yingakho izinto eziguquguqukayo zenqubo — ukugxila kukadayi, i-buffer, isilinganiso segazi nodayi, izinga lokushisa nesikhathi — zibaluleke njengegama likadayi.
Umphumela walokhu ekusebenzeni ukuhleleka okuqinile kwezinyathelo: qala ngokuxuba igazi elifakwe into evimbela ukujiya nodayi, uliyeke isikhathi esibekiwe ezimweni ezifanele, bese ulisatshalalisa libe izingqimba ezincane futhi ulihlole lingafiksiwe futhi lingafakwanga udayi wesibili. Ukufiksa ngaphambi kokudaya, noma ukusebenzisa udayi wesibili ositha imicu engabonakali kahle, kuqeda ulwazi ukubala okuncike kulo.
Inqubo esebenzayo yama-reticulocyte nge-new methylene blue
Akukho ukwakheka kwesixazululo okukodwa okusetshenziswa yonke indawo, ngakho bika lokho okusetshenzisiwe. Izindlela ezintathu ezibhalwe phansi zikhombisa ububanzi bokwahluka.
Inqubo yakudala yohlobo lwe-Brecher isebenzisa cishe i-new methylene blue engu-0.5 percent ene-potassium oxalate, ixutshwe ngevolumu elinganayo negazi le-EDTA elixutshwe kahle, bese iyekwa cishe imizuzu engu-10 kuya kwengu-15 ezingeni lokushisa legumbi. Ngemva kokuyixuba kabusha ngobumnene, kwenziwa izingqimba ezincane ezimise okwe-wedge, zomiswe emoyeni, bese zihlolwa zingafiksiwe ngelensi ye-objective engu-100x esebenzisa uwoyela. Inqubo ye-NCBI yokubala ama-reticulocyte ichaza lokhu kuhamba komsebenzi nohlaka lokubala olusetshenziswa nalo.
Indlela ye-Dacie no-Lewis isebenzisa i-new methylene blue engu-1.0 percent esixazululweni sikasawoti esine-citrate, ixutshwe negazi eliyivolumu ephindwe ka-2 kuya ku-4 kuneyodayi bese igcinwa ku-37 C imizuzu engu-15 kuya kwengu-20 ngaphambi kokusabalalisa amasampula abe izingqimba ezincane. Ukusetshenziswa kwakamuva kwenqubo ye-CLSI H44 yokuvumelanisa izindlela kwasebenzisa isixazululo esingagxilile kangako esingu-0.1 percent esixazululweni sikasawoti esine-phosphate buffer ku-pH 7.4, sagcinwa ku-20 C imizuzu engu-10 futhi sangafikswa, njengoba kubikiwe ocwaningweni luka-2026 lokuvumelanisa i-NMB.
Imithetho yokubala iyafana kuzo zonke lezi zindlela. Bala iseli njenge-reticulocyte uma linezinhlayiyana ezidaywe zaluhlaza okwesibhakabhaka noma izingxenye zemicu okungenani ezimbili ezihlukene, okungafaki idiphozithi kadayi eyodwa ezimele. Bala okungenani amaseli abomvu egazi angu-1,000 emsebenzini ojwayelekile bese ubika ama-reticulocyte njengephesenti. Lapho amaphesenti ephansi, izibalo zidinga amaseli amaningi. Ukubuyekezwa kwendlela ye-CLSI H44 kuphawula ukuthi uma ama-reticulocyte ecishe abe ngu-1 percent, kudingeka cishe amaseli angu-4,400 ukuze ukwahluka kokubala kwehle kufinyelele ku-5 percent, ekubuyekezweni kwe-CLSI H44 ku-De Gruyter. Idiski ye-Miller efakwa engxenyeni yesibonakhulu yokubuka ngeso yenza kube lula ukubala amaseli amaningi. Hlunga udayi noma uwulungise usha, ngoba izinsalela zodayi ophume esixazululweni zifana nezinhlayiyana ezingaphakathi kwamaseli.
Imizimba ka-Heinz esampuleni efanayo
Ungqimba olufanayo oludaywe nge-supravital lungakhombisa nemizimba ka-Heinz, okuyizinhlanganisela ze-hemoglobin eyenziwe i-oxidation futhi esiguquke ekwakhekeni kwayo, hhayi i-RNA eyinsalela. Kumasampula e-new methylene blue, ibonakala njengezakhi eziyindilinga, ngokuvamile iyodwa futhi iseduze kolwelwesi lweseli, idaywe ngombala oluhlaza okwesibhakabhaka okhanyayo kunokwakheka kwenethiwekhi nemicu. Ayilondolozeki kahle lapho kufikswa futhi kufakwa udayi wesibili, ngakho hlola ungqimba olungafiksiwe. Inqubo yakamuva esebenzisa ama-microliter angu-20 egazi le-EDTA nama-microliter angu-20 e-new methylene blue engu-1 percent, kanye nokuyeka ingxube imizuzu engu-15, yabala amaseli anemizimba ka-Heinz phakathi kwama-erythrocyte angu-250, ocwaningweni lwe-PMC olubika leyo nqubo.
Ukuhlukanisa izinto ezimbili kuvimbela ukuhumusha okungalungile. Okokuqala, i-methyl violet ne-crystal violet zidaya imizimba ka-Heinz ngokugqamile kakhulu kune-new methylene blue, njengoba kuphawuliwe esihlokweni se-ScienceDirect ngemizimba ka-Heinz. I-new methylene blue iyindlela elula uma ama-reticulocyte nemizimba ka-Heinz kuhlolwa ndawonye, kodwa ayiwona udayi oqine kakhulu owenzelwe imizimba ka-Heinz kuphela. Okwesibili, izakhi ze-HbH ezingaphakathi kwamaseli kubantu abanofuzo lwe-alpha thalassemia zenza amachashaza amaningi amancane kulo lonke iseli ngemva kokudaya nge-supravital, kuyilapho imizimba ka-Heinz ejwayelekile imbalwa, imikhulu, futhi ivame ukuhlobana nolwelwesi lweseli. Idiphozithi kadayi iyinto yesithathu efana nazo: ayikho ezingeni elifanayo lokugxila kwesibonakhulu namaseli futhi iyahluka ezindaweni ezibukwayo.
Ukuqhathaniswa kwe-brilliant cresyl blue ne-new methylene blue
Bobabili lab' odayi bangodayi be-supravital bama-reticulocyte, kodwa ukubekwa kwezindlela ngokwezinga elifanayo kuvuna oyedwa wabo. Indlela ye-CLSI H44-A2 yama-reticulocyte, eyakhiwe ngokubambisana ne-International Council for Standardization in Haematology futhi esohlwini ekhasini lezinga le-CLSI H44, ibeka ukudaya igazi le-EDTA nge-new methylene blue njengendlela yokusebenza ngesandla esetshenziselwa ukuqhathanisa. Imfundiso yakudala yocwaningo lwegazi iyavumelana ngendlela okubonakala ngayo: i-new methylene blue idaya imicu ngokujulile nangokulinganayo kakhulu, kuyilapho i-brilliant cresyl blue ihluka kakhulu phakathi kwamaqoqo odayi namasampula.
Leso sikhundla sidinga umbandela waso. Umhlahlandlela we-ICSH wokuhlola izihlaziyi wamukela ukuhlola ngesandla ngaphansi kwesibonakhulu kusetshenziswa i-new methylene blue njengendlela yokuqhathanisa eyinkomba, kodwa uthi kuncike ekwahluleleni komuntu futhi ukunemba kwayo kulinganiselwe; izinqubo eziyinkomba ze-flow cytometry zikhethwa lapho zitholakala khona. Ukuqhathaniswa kwamasampula angu-100 ngo-2022 kwathola ukuchezuka okuncane emiphumeleni yesihlaziyi esisetshenzisiwe nge-brilliant cresyl blue kunange-new methylene blue, njengoba kubikiwe ekuqhathanisweni kwezindlela zokudaya ku-PMC. Isifinyezo esinobufakazi esinganikwa abafundi sithi: sebenzisa i-new methylene blue njengendlela yokusebenza ngesandla yokubhekisa ekuqhathaniseni ebekwe ngokwezinga elifanayo, qinisekisa noma iyiphi indlela yendawo ye-brilliant cresyl blue ngokuyiqhathanisa nayo noma nohlelo oluzenzakalelayo, futhi ulindele ukuhlakazeka okubanzi kwemiphumela ekuhloleni ngesandla ngaphansi kwesibonakhulu kungakhathaliseki ukuthi ukhetha muphi udayi. Izincwadi ezindala zokubeka izindlela ngokwezinga elifanayo nazo zakhuthaza i-Azure B ehlanziwe njengenye indlela enokwakheka kwamakhemikhali okuhlanzeke kakhulu ngoba amaqoqo odayi abathengiswayo ayehluka, ocwaningweni luka-Marshall nge-Azure B, okuchaza ukuthi kungani izinga lobumsulwa be-reagent lisavela ezingxoxweni zezindlela.
Ukugwema amaphutha ajwayelekile
Amaphutha amane abangela ukwehluleka kwamasampula amaningi alungisiwe. Uku-oda i-methylene blue evamile esikhundleni se-new methylene blue N kubangela ukudaya okubuthakathaka ngenxa yekhemistri, hhayi indlela yokusebenza. Ukufiksa noma ukufaka udayi wesibili ngaphambi kokuhlola kususa lokho okuboniswa ukudaya nge-supravital. Ukubala izinsalela zodayi ophume esixazululweni njengezinhlayiyana kwenyusa umphumela ngokungafanele; umthetho wezinhlayiyana ezimbili nokusebenzisa udayi ohlungiwe kuyakuvimbela lokho. Ukubala amaseli ambalwa kakhulu lapho amaphesenti ama-reticulocyte ephansi kukhiqiza inombolo ebonakala inembile kodwa enemikhawulo ebanzi yezinga lokuqiniseka. Iphutha ngalinye linophawu olufanayo: osebenzisa isibonakhulu usola isibonakhulu ngalokho okwanqunywa yisinyathelo se-reagent.