Methylene blue a gwaje-gwajen G6PD na ɗakin gwaje-gwaje: ainihin abin da rinin yake yi

Wani ɗalibi yana kallon bututun gwaji biyu bayan an bar su a yanayin zafin da aka tanada. Ɗaya ya koma launin da ake tsammani. Ɗayan bai koma ba. Ɗalibin ya yanke cewa majiyyaci na biyu yana da ƙarancin glucose-6-phosphate dehydrogenase. Wannan hukuncin na iya zama daidai, amma hanyar tunanin ta tsallake muhimmin abin da ke sa gwajin aiki. Babu wani abu a cikin bututun da ke auna enzayim ɗin kai tsaye. Bututun yana auna abin da enzayim ɗin ke samarwa, yana amfani da methylene blue a matsayin mai shiga tsakani.

Da zarar an fahimci wannan aunawa ta hanyar abin da enzayim ɗin ke samarwa, matakan gwaji, samfurorin kwatanci, da jerin dalilan sakamakon ƙarya ba sa ƙara zama kamar an zaɓe su ba tare da dalili ba.

Amsa a taƙaice

Methylene blue a gwajin G6PD alamar redox ce. Enzayim ɗin G6PD yana samar da NADPH, kuma tsarin da ke dogara da NADPH yana yin ridakshan na methylene blue mai shuɗi zuwa sigarsa ta leuco marar launi (ko kuma yana amfani da shi wajen jigilar electron). Aikin enzayim na al'ada yana samar da ridakshan mai inganci. Idan aikin ya yi ƙasa, ridakshan ɗin yana tafiya a hankali ko kuma ba ya cika. Wannan ne duk abin da alamar ke nunawa.

Hanyoyi biyu na tarihi suna amfani da rini ɗaya, amma suna da bambancin abin da ake dubawa a ƙarshen gwaji. Idan aka rarrabe su, yawancin ruɗanin zai gushe.

Gwaje-gwaje biyu, rini ɗaya, alamomin ƙarshe daban-daban

Na farko shi ne gwajin ridakshan na methylene blue kai tsaye, wanda Sass da abokan aikinsa suka bayyana, sannan Gibbs ya tantance shi daga baya. A nan, alamar ƙarshe ita ce rinin kansa: ana yin ridakshan na methylene blue mai shuɗi da aka yi wa oksidashan zuwa leucomethylene blue marar launi, kuma saurin wannan sauyi yana nuna ƙarfin samar da NADPH. An jera takardar asali ta gwajin tantancewar a PubMed a ƙarƙashin Sass da abokan aikinsa (1966).

Na biyu shi ne gwajin ridakshan na methemoglobin na Brewer. Da farko, sodium nitrite yana yin oksidashan na hemoglobin zuwa methemoglobin mai launin ruwan kasa. Sai methylene blue ya zama mai shiga tsakani wajen canja wurin electron: methylene blue da aka yi wa ridakshan yana miƙa abubuwan da ke ba da damar ridakshan zuwa methemoglobin, yana mai da shi hemoglobin mai ja. Saboda haka, alamar ƙarshe da ido ke gani ita ce ruwan kasa idan aka kwatanta da ja, ba shuɗi idan aka kwatanta da rashin launi ba. Hanyar asali ta bayyana a cikin Bulletin of the World Health Organization (Brewer da abokan aikinsa, 1960).

Kiran gwajin Brewer gwajin cire launin rini ba shi da cikakken daidaito a fannin sinadarai. Rinin yana shiga zagayen sauye-sauye; hemoglobin ne yake sauya launi. Zanen da ke ƙasa yana nuna hanyoyin biyu.

Mu'amalar sinadarai da gwajin ya dogara da ita

G6PD yana hanzarta matakin oksidashan na farko a hanyar pentose phosphate:

Glucose-6-phosphate + NADP+ becomes 6-phosphogluconolactone + NADPH + H+

Balagaggun jajayen ƙwayoyin jini suna dogara sosai da wannan hanyar domin samun NADPH, kamar yadda aka yi nazari a Blood ta Luzzatto da abokan aikinsa (2020). NADPH yana riƙe glutathione a yanayin da aka yi wa ridakshan, kuma yana kare ƙwayar halitta daga lalacewar oksidashan. Methylene blue yana shiga wannan tsarin a matsayin mai karɓar electron. Isasshen G6PD yana samar da isasshen NADPH, wanda ke samar da ridakshan mai inganci. Ƙarancin G6PD yana katse wannan jerin tun daga matakinsa na farko.

Ka ɗauki rinin a matsayin ma'aunin man fetur, ba injin ba. Yana nuna wadatar NADPH. Duk wani abu da ya sauya wannan wadatar, ko yadda ake karanta sakamakon, zai iya sauya abin da ma'aunin ke nunawa ba tare da wani sauyi a kwayar gado ba.

Yadda ake gudanar da gwaji irin na Brewer

Haɗin sinadaran gwaji ya bambanta a tsawon shekarun da suka gabata, don haka bai kamata a ɗauki bayanin koyarwa a matsayin ƙa'idar gwajin asibiti ba. Kowane ɗakin gwaje-gwaje yana bin nasa ingantaccen tsarin aiki. Da wannan gargaɗin, tsarin bututun gwaji na gargajiya yana aiki kamar haka, a sigar da aka rubuta da bututu uku da barinsu a zafin da aka tanada na tsawon awa 3 a wannan aiwatarwar da aka wallafa:

1. Bututun majiyyaci: jini cikakke tare da glucose, sodium nitrite, da methylene blue. Nitrite yana samar da methemoglobin, glucose yana tallafa wa tsarin sinadaran jajayen ƙwayoyin jini, kuma rinin yana samar da hanyar jigilar electron da ke dogara da NADPH.
2. Samfurin kwatanci da aka yi wa oksidashan: jini tare da nitrite da glucose amma babu methylene blue. Yana nuna alamar ƙarshe ta methemoglobin mai ruwan kasa, wato yanayin ƙaranci.
3. Samfurin kwatanci na hemoglobin: jini tare da ruwa. Yana nuna alamar ƙarshe ta hemoglobin mai ja, wato yanayin al'ada.
4. A bar duk bututun a zafin da ke kusa da 37 C, yawanci na tsawon awa 3, ana gaurayawa a hankali.
5. A tsarma ɗan kaso daga kowane bututu cikin ruwan da aka tace ta tururi domin a tarwatsa ƙwayoyin jinin kuma a sauƙaƙa kwatanta launuka.
6. A kwatanta bututun majiyyaci da samfurorin kwatanci biyu. Komawa wajen launin ja yana nuna ƙarfi mai yawa na yin ridakshan. Ci gaba da kasancewa kusa da ruwan kasa yana nuna yiwuwar ƙaranci mai tsanani.

Wani haɗin tarihi ya yi amfani da kusan 1 mL na jini tare da sinadaran gwaji masu kusan 180 mM sodium nitrite, 280 mM dextrose, da 0.4 mM methylene blue. A ɗauki waɗannan adadin a matsayin misali daga wallafe-wallafen kimiyya, ba ƙa'idar da ta shafi ko'ina ba.

Karanta sakamakon

Gwajin tantancewa da ba ya bayar da adadi yana bambance yanayi mafi kyau a kusan kashi 30 zuwa 40 na aikin al'ada, kamar yadda aka tattauna a wannan nazarin Malaria Journal kan giɓin gano G6PD. Saboda haka, sakamakon tantancewa na al'ada yana nufin gwajin bai gano ƙaranci mai tsanani ba. Ba ya tabbatar da cewa aikin enzayim ɗin yana daidai da al'ada gaba ɗaya.

Dalilin da ya sa tantancewa kan ruɗar

Ƙaruwa na reticulocytes bayan tarwatsewar jajayen ƙwayoyin jini shi ne sanannen dalilin sakamakon al'ada na ƙarya. Matasa jajayen ƙwayoyin jini da reticulocytes suna ɗauke da G6PD fiye da tsofaffin ƙwayoyin. A lokacin tarwatsewar jajayen ƙwayoyin jini mai tsanani, mai yiwuwa an riga an lalata ƙwayoyin da suka fi tsufa kuma suka fi ƙarancin G6PD, a bar tarin matasan ƙwayoyin da aikinsu ke fitowa a matsayin al'ada a gwaji. Wannan yanayin na halitta yana ruɗar da gwaje-gwajen ƙididdigar adadi ma, ba gwaje-gwajen tantancewa na rini kaɗai ba.

Ƙarin jini da aka yi kwanan nan yana ɓoye ƙaranci saboda jajayen ƙwayoyin jinin mai bayarwa suna ƙara nasu enzayim cikin samfurin. Mayo Clinic Laboratories ya jera ƙarin jini cikin dalilan sakamakon enzayim marasa tabbas, tare da ƙaruwar reticulocytes da katsalandan daga fararen ƙwayoyin jini.

Mata masu sigogi biyu daban-daban na kwayar gado su ne mafi wahalar tantancewa. Kashe aikin chromosome X ba tare da takamaiman tsari ba yana haifar da cakuduwar jajayen ƙwayoyin jini na al'ada da masu ƙaranci, kuma jimillar aikin na iya kasancewa a ko'ina daga ƙaranci mai tsanani zuwa abin da ke kama da al'ada. Gwaje-gwajen tantancewa da ba sa bayar da adadi sukan kasa gano yanayin tsaka-tsaki. Takardun WHO kan gwajin G6PD sun tattauna wannan iyaka dalla-dalla.

Tsawon lokacin da samfurin ya yi, zafin adanawa, bambancin ra'ayi wajen tantance alamar ƙarshe da ido, hematocrit, da ingancin sinadaran gwaji suna ƙara sauye-sauyen da ba a so a bincike, ban da waɗannan tasirin halitta.

Tantancewa ba tabbatar da ganewar yanayi ba ce

Sakamakon tantancewa da ya nuna ƙaranci, tsaka-tsaki, ko wanda bai dace da alamun da ake gani a majiyyaci ba yana buƙatar gwajin ƙididdigar aikin enzayim ɗin G6PD. WHO tana ɗaukar spectrophotometry na ƙididdigar adadi da ake sarrafa zafinsa a matsayin hanyar kwatanci. A wannan gwajin, ana ƙara glucose-6-phosphate da NADP+ cikin ruwan da aka samu daga tarwatsa jajayen ƙwayoyin jini, sannan ana bibiyar NADPH da ya samu ta hanyar ƙaruwar shan haske a 340 nm. Saurin sauyin yana tafiya daidai da aikin enzayim, kuma yawanci ana daidaita shi bisa yawan hemoglobin a matsayin raka'o'i a kowane gram. An taƙaita tsarin da ɗakunan gwaje-gwajen asibiti ke bi a yanzu a bayanin kundin gwajin Mayo na aikin G6PD.

Fassarar sakamako tana amfani da zangon kwatanci da ɗakin gwaje-gwaje ya tabbatar da ingancinsa, wanda galibi ake bayyana shi dangane da aikin al'ada a cikin jama'a, tare da tattauna ƙaranci a kusa da iyakar kashi 30 da aikin tsaka-tsaki sama da ita. Bayanan WHO na halayen samfuran gwajin G6PD da ake nema (2022) sun fayyace wannan tsarin ganewar yanayi. Idan tarwatsewar jajayen ƙwayoyin jini ko ƙarin jini da aka yi kwanan nan ya sa tantance yanayin da ya bayyana ba ya da tabbas, a maimaita gwaji bayan tarin jajayen ƙwayoyin jini ya sake daidaituwa, kuma gwajin ƙwayoyin halitta na iya ƙara bayani ba tare da maye gurbin auna enzayim ba.

Bambancin da ya kamata a kiyaye

Methylene blue a waɗannan gwaje-gwajen sinadarin gwaji ne da ake amfani da shi a wajen jiki. Wannan batu ya bambanta da tattaunawar amincin ba majiyyaci methylene blue, wadda aka yi bayani a jagora kan yanayin da bai kamata a yi amfani da methylene blue ba. Ilimin sinadaran halitta yana haɗa batutuwan biyu, domin maganin ma yana dogara da ridakshan da ke buƙatar NADPH, amma rini a cikin bututun gwaji ba adadin magani ne a cikin jini ba. Don ƙarin bayani kan yadda rinin ke aiki wajen yin tabo a wani yanayi dabam, duba ƙa'idodin yin tabo da methylene blue, sannan don rukunin sinadaran da yake ciki, rinayen redox a ɗakin gwaje-gwaje.

Tantancewa tana tambayar ko ƙarfin ridakshan yana kama da wanda bai samu tangarɗa ba. Tabbatarwa tana tambayar adadin enzayim da yake nan a zahiri. Rinin yana amsa tambaya ta farko da kyau, matuƙar ba a nemi ya amsa ta biyu ba.