New methylene blue da rina reticulocyte: rini na daban da hanyar ƙwayoyin halitta masu rai

Wani ɗalibi yana shirin ƙidayar reticulocyte, ya yi odar methylene blue, ya haɗa shi da jini, amma bai ga kusan komai ba. Abokin karatunsa ya yi amfani da kwalbar da aka yi wa lakabin new methylene blue, kuma ya ga zaren shuɗi masu bayyana sarai a cikin wani ɓangare na jajayen ƙwayoyin jini. Ɗalibin farko ya ɗauka cewa rinin kwalba ta biyu ya fi sabo ko ya fi ƙarfi. Ba ɗaya daga cikin waɗannan ba ne. Ya ƙunshi wata kwayar sinadari dabam.

Wannan kuskuren fahimta shi ne tushen mafi yawan ruɗanin wannan fanni. Sunan yana sa a ɗauka cewa new methylene blue wani ingantaccen rukuni ne na methylene blue. Amma ilimin sinadarai ya nuna ba haka ba ne. New methylene blue N wani rinin phenothiazine ne na daban, mai lambar Colour Index tasa, tsarin ƙungiyoyin sinadarai da suka maye gurbin wasu a tsarinsa, da takamaiman aikin da aka daidaita a ilimin jini. Da zarar an fahimci wannan bambanci, sauran hanyar aikin za ta zama mai sauƙin fahimta: dalilin da ya sa dole ƙwayoyin su kasance da rai lokacin rina su, abin da ake ƙidaya a matsayin reticulocyte, matsayin jikin Heinz, da lokacin da brilliant cresyl blue yake shiga cikin aikin.

Amsa a taƙaice

New methylene blue N, sinadarin aikin ilimin jini, yawanci ana gane shi a matsayin Basic Blue 24 a bayanan PubChem na new methylene blue, C.I. 52030, CAS 1934-16-3, mai dabarar sinadarai C18H22ClN3S. Methylene blue na yau da kullum shi ne methylthioninium chloride, Basic Blue 9, a bayanansa na PubChem, C.I. 52015, CAS 61-73-4, mai dabarar sinadarai C16H18ClN3S. A tsarin kwayar sinadarinsa, new methylene blue yana ɗauke da ƙungiyoyin methyl biyu a zoben sinadarin da rassan ethylamino a wuraren da methylene blue yake ɗauke da ƙungiyoyin dimethylamino. Wannan bambanci yana sauya yadda yake rina abubuwa. Littattafan ilimin jini suna bayyana methylene blue na yau da kullum a matsayin rinin da bai ƙware sosai wajen rina reticulocyte ba, yayin da new methylene blue yake ba da rini mai zurfi da daidaito; an taƙaita wannan kwatanci a batun new methylene blue na ScienceDirect.

Eh, new methylene blue rini ne na supravital. Rina ta supravital tana nufin rina ƙwayoyin halitta masu rai bayan an cire su daga jiki, amma kafin a tsayar da su da sinadari, kamar yadda aka bayyana a bitar NCBI kan ƙa'idojin rina ta supravital. New methylene blue da brilliant cresyl blue duk suna cikin wannan rukuni, kuma ma'aunin CLSI na reticulocyte ya bayyana new methylene blue kai tsaye a matsayin rini na supravital. Wani lokaci ɗalibai suna haɗa wannan da rina ta Romanowsky a shafukan jini da aka riga aka tsayar da sinadari. Wannan fahimtar ba ta dace a nan ba. Idan an fara da tsayar da ƙwayoyin da sinadari, hanyar ba za ta yi aiki ba.

Ga masu bibiyar sunayen rinaye masu kama da juna, bambancin methyl blue da methylene blue yana bayani kan wani ruɗani na daban kuma mafi girma tsakanin rinin triarylmethane mai cajin korau da dangin phenothiazine masu cajin tabbatacce. Bayani na gaba ɗaya kan rina a dakin gwaje-gwaje yana nuna matsayin aikin supravital a tsakanin hanyoyin amfani da shafukan da aka tsayar da sinadari, kuma jagorar siffar ƙwayoyin jini tana nuna inda tantance reticulocyte yake a cikin faɗaɗɗen aikin bambance nau'ikan ƙwayoyin jini.

Gargaɗi guda game da kundin kayayyaki: akwai wani nau'in new methylene blue na gishiri biyu mai zinc chloride a ƙarƙashin CAS 6586-05-6. Saboda haka, sashen hanyoyin aiki ya kamata ya ambaci nau'in gishirin da mai samarwa, ba sunan da aka saba amfani da shi kaɗai ba.

Abin da rina ta supravital take yi a zahiri

Reticulocyte matashiyar jajayen ƙwayar jini ce wadda ta rasa tsakiya, amma har yanzu tana ɗauke da ragowar RNA na ribosome. A samfurin jika da ba a rina ba, wannan RNA ba ya bayyana. Rinin supravital yana shiga ƙwayar mai rai, sannan ya sa wannan RNA ya taru ko ya haɗu zuwa ƙananan ɗigon shuɗi da zare, wato kayan da suka yi kama da raga da zare da ake gani lokacin dubawa da man immersion. Saboda haka, wannan ragar sakamakon rina ce, ba wani tsarin da ya riga ya kasance yana jiran a ba shi launi ba. Wannan shi ya sa abubuwan da suke sauyawa a tsarin aiki — ƙarfin rinin, sinadarin daidaita pH, rabon jini da rini, zafin jiki, da lokaci — suke da muhimmanci kamar sunan rinin.

Sakamakon wannan a aikace shi ne bin tsari mai tsauri: da farko a haɗa jinin da aka hana daskarewa da rini, a bar shi na lokacin jira da aka tsara, sannan a yi shafuka a duba su ba tare da tsayar da su da sinadari ko ƙara rini na biyu ba. Tsayar da ƙwayoyin da sinadari kafin rina su, ko ƙara rini na biyu da zai ɓoye zaren da ba su bayyana sosai ba, yana lalata bayanin da ƙidayar ta dogara da shi.

Tsarin aiki na ƙidayar reticulocyte da new methylene blue

Babu haɗi guda ɗaya da kowa yake amfani da shi, don haka a bayyana wanda aka yi amfani da shi. Hanyoyi uku da aka rubuta suna nuna irin bambancin da ake da shi.

Hanyar gargajiya ta nau'in Brecher tana amfani da kusan 0.5 bisa ɗari na new methylene blue tare da potassium oxalate. Ana haɗa shi da jinin EDTA da aka gauraya sosai a ma'aunin ƙarar da ya yi daidai, sannan a bar shi kusan mintuna 10 zuwa 15 a zafin ɗaki. Bayan an sake gaurayawa a hankali, ana yin siraran shafuka masu siffar tsinin alƙalami, a bar su su bushe a iska, sannan a duba su ba tare da tsayar da su da sinadari ba, da ruwan tabarau na kusa da samfur mai 100x da ake amfani da shi da mai. Hanyar NCBI ta ƙidayar reticulocyte tana bayyana wannan jerin aikin da firam ɗin ƙidaya da ake amfani da shi.

Hanyar Dacie da Lewis tana amfani da 1.0 bisa ɗari na new methylene blue a ruwan gishiri mai citrate, ana haɗa shi da jini mai ƙara sau 2 zuwa 4, sannan a ajiye shi a 37 C na mintuna 15 zuwa 20 kafin yin shafuka. Wani aiwatarwa na baya-bayan nan don daidaita hanyar CLSI H44 ya yi amfani da magani mai ƙarancin ƙarfi, wato 0.1 bisa ɗari a ruwan gishiri mai phosphate da ke daidaita pH zuwa 7.4, tare da jira a 20 C na mintuna 10 kuma ba tare da tsayar da ƙwayoyin da sinadari ba, kamar yadda aka ruwaito a binciken daidaita NMB na 2026.

Ƙa'idojin ƙidaya iri ɗaya ne a duk waɗannan hanyoyin. A ƙidaya ƙwaya a matsayin reticulocyte idan tana ɗauke da aƙalla ƙananan ɗigon shuɗi ko sassan zare guda biyu da suka rabu da juna; hakan yana ware dunƙulen rini guda ɗaya da ya sauka shi kaɗai. A ƙidaya aƙalla jajayen ƙwayoyin jini 1,000 a aikin yau da kullum, sannan a bayar da adadin reticulocyte a matsayin kaso bisa ɗari. Idan kaso ya yi ƙasa, ƙa'idojin ƙididdiga suna buƙatar a ƙidaya ƙwayoyi masu yawa. Wata bita kan hanyar CLSI H44 ta nuna cewa, idan reticulocyte suna kusan 1 bisa ɗari, ana buƙatar kusan ƙwayoyi 4,400 domin rage bambancin ƙidaya zuwa 5 bisa ɗari, a bitar CLSI H44 a De Gruyter. Faifan Miller da ake saka a ruwan tabarau na ido yana sauƙaƙa yin manyan ƙidaya. A tace rinin ko a shirya shi sabo, domin rinin da ya taru ya fita daga magani yana kama da ƙananan ɗigon abubuwa a cikin ƙwayoyin.

Jikin Heinz a cikin wannan samfurin

Wannan shafin supravital ɗin zai iya nuna jikin Heinz, waɗanda su ne tarin hemoglobin da ya yi oxidation kuma ya rasa tsarin halittarsa, ba ragowar RNA ba. A samfuran new methylene blue, suna bayyana a matsayin abubuwa masu zagaye, sau da yawa guda ɗaya kuma kusa da fatar ƙwayar, suna ɗaukar shuɗi mai haske fiye da kayan da suka yi kama da raga da zare. Ba sa dawwama sosai bayan tsayar da ƙwayoyin da sinadari da ƙara rini na biyu, don haka a duba shafin da ba a tsayar da sinadari ba. Wani tsarin aiki na baya-bayan nan da ya yi amfani da 20 microliters na jinin EDTA tare da 20 microliters na new methylene blue mai 1 bisa ɗari da lokacin jira na mintuna 15 ya ƙidaya ƙwayoyin da suka nuna jikin Heinz a cikin jajayen ƙwayoyin jini 250, a binciken PMC da ya ruwaito wannan hanyar.

Bambance-bambance biyu suna hana kuskuren fassara abin da aka gani. Na farko, methyl violet da crystal violet suna rina jikin Heinz da ƙarfi fiye da new methylene blue, kamar yadda aka lura a batun jikin Heinz na ScienceDirect. New methylene blue zaɓi ne mai sauƙin amfani idan ana tantance reticulocyte da jikin Heinz tare, amma ba shi ne rini mafi ƙarfi da aka keɓe domin jikin Heinz ba. Na biyu, tarin HbH a cikin ƙwayoyin masu ɗauke da halin alpha thalassemia yana samar da ƙananan ɗigo da yawa a duk faɗin ƙwayar bayan rina ta supravital, yayin da jikin Heinz na gargajiya suka fi ƙarancin yawa, suka fi girma, kuma sau da yawa suna kusa da fatar ƙwayar. Dunƙulen rinin da ya sauka shi ne abu na uku da zai iya yin kama da su: yana wajen matakin da ƙwayoyin suke bayyana sarai a madubin, kuma yana bambanta daga wani filin dubawa zuwa wani.

Brilliant cresyl blue idan aka kwatanta da new methylene blue

Duk rinayen biyu rinaye ne na supravital don reticulocyte, amma ƙa'idojin daidaitawa suna fifita ɗaya daga cikinsu. Hanyar CLSI H44-A2 ta reticulocyte, wadda aka haɓaka tare da Majalisar Ƙasa da Ƙasa ta Daidaita Ayyuka a Ilimin Jini kuma aka jera a shafin ma'aunin CLSI H44, ta ayyana rina jinin EDTA da new methylene blue a matsayin hanyar kwatanci ta aikin hannu. Koyarwar gargajiya ta ilimin jini ta yarda da yadda suke bayyana: new methylene blue yana ba da rina zare mai zurfi da daidaito, yayin da brilliant cresyl blue yake nuna bambanci mafi yawa tsakanin rukunin samarwa da samfura.

Wannan matsayi yana buƙatar ƙarin bayani. Jagorar ICSH ta tantance na'urorin nazari ta amince da dubawa da madubin ƙwayoyin halitta ta amfani da new methylene blue a aikin hannu a matsayin hanyar kwatanci da ma'aunin tunani, amma ta bayyana cewa tana dogara da hukuncin mai dubawa kuma ba ta da babban daidaiton ƙidaya. An fi son hanyoyin ma'aunin tunani na flow cytometry idan suna samuwa. Wani kwatanci na samfura 100 a 2022 ya gano cewa brilliant cresyl blue ya nuna karkacewa mafi ƙanƙanta daga sakamakon na'urar nazarinsa fiye da new methylene blue, kamar yadda aka ruwaito a kwatancin hanyoyin rina na PMC. Taƙaitaccen bayani mai hujja ga ɗalibai shi ne: a yi amfani da new methylene blue a matsayin ma'aunin kwatanci na aikin hannu da aka daidaita, a tabbatar da kowace hanyar brilliant cresyl blue ta wurin aiki ta hanyar kwatanta ta da shi ko da na'ura, kuma a sa ran sakamakon dubawa da hannu zai fi warwatse a kowace hanya. Tsofaffin wallafe-wallafen daidaitawa sun kuma ba da shawarar Azure B da aka tsarkake a matsayin madadin da ya fi tsabta ta fuskar sinadarai, domin rukunin rinayen kasuwanci suna bambanta, a binciken Marshall kan Azure B. Wannan yana bayyana dalilin da ya sa har yanzu ake tattauna matsayin ingancin sinadarin aiki a bayanan hanyoyi.

Guje wa kurakuran da aka saba yi

Kurakurai huɗu ne ke haddasa yawancin gazawar shirya samfura. Yin odar methylene blue na yau da kullum maimakon new methylene blue N yana haifar da rini mara ƙarfi saboda yanayin sinadarin, ba saboda dabarar aiki ba. Tsayar da ƙwayoyin da sinadari ko ƙara rini na biyu kafin dubawa yana kawar da abin da rina ta supravital take nunawa. Ƙidaya dunƙulen rinin da ya fita daga magani a matsayin ƙananan ɗigo yana ƙara sakamakon fiye da gaskiya; ƙa'idar ɗigo biyu da tace rini suna hana hakan. Ƙidaya ƙwayoyi kaɗan idan kaso na reticulocyte ya yi ƙasa yana samar da lamba mai kama da ingantacciya, amma mai faɗin iyakokin amincewar ƙididdiga. Kowanne kuskure yana da alama iri ɗaya: mai amfani da madubin ƙwayoyin halitta yana ɗora laifi a kan madubin saboda abin da matakin amfani da sinadarin aiki ya riga ya ƙayyade.