Pjautuvinës anemijos laboratorinë diagnostika
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1 MOKSLO DARBAI SCIENTIFIC PAPERS Laboratorinë medicina. 2009, t. 11, Nr. 1(41), p Pjautuvinës anemijos laboratorinë diagnostika Rëda Matuzevièienë 1, 2 Asta Maèionienë 1, 3 1 Vilniaus universiteto Medicinos fakulteto fiziologijos, biochemijos ir laboratorinës medicinos katedra, M. K. Èiurlionio g. 21/27, LT Vilnius Department of Physiology, Biochemistry and Laboratory Medicine, Faculty of Medicine, Vilnius University, M.K. Èiurlionio str. 21/27, LT Vilnius, Lithuania 2 Vilniaus universiteto ligoninës Santariðkiø klinikø Laboratorinës diagnostikos centro Hematologijos ir bendraklinikiniø tyrimø laboratorija El. paðtas: reda.matuzeviciene@santa.lt Vilnius University Hospital Santariðkiø Clinics Laboratory of Haematology and Cytology, Santariðkiø str. 2, Vilnius, Lithuania reda.matuzeviciene@santa.lt 3 Vilniaus universiteto ligoninës Santariðkiø klinikø Laboratorinës diagnostikos centro Mikrobiologijos laboratorija, Santariðkiø g. 2, LT Vilnius El. paðtas: asta.macioniene@santa.lt Vilnius University Hospital Santariðkiø Clinics Laboratory of Microbiology Santariðkiø str. 2, LT Vilnius, Lithuania asta.macioniene@santa.lt Santrauka Pjau tu vi në ane mi ja, dar va di na ma pjau tu vi niø làs te liø li ga, pri ski ria - ma paveldimø struktûriniø hemoglobinopatijø grupei. Tai autosominiu re ce sy vi niu bû du pa vel di ma li ga, ku rià su ke lia tað ki në mu ta ci ja glo bi no gene. Dël ðios mutacijos susidaræs struktûriðkai nenormalus hemoglo - bi no (Hb S) tet ra me ras de ok si ge na ci jos sà ly go mis lin kæs po li me ri zuo tis. Dël struktûriniø ir funkciniø eritrocito membranos pokyèiø ðie ágyja pjautuvo formà ir uþkemða smulkiàsias kraujagysles, greièiau hemoli - zuo ja si. Krau ja gys liø ok liu zi ja su ke lia au di niø ið emi jà ir skaus mi nes kri zes. Lë ti në ane mi ja ir au di niø ið emi ja lemia ðir dies, plau èiø, ðla pi mo or ga nø sis te mos, ske le to, re gos, odos pa þei di mus, neu ro lo gi næ simp to - ma ti kà. Ga li ma sun ki li gos kom pli ka ci ja au to sple nek to mi ja. Dël ðios prie þas ties li go niø im lu mas in fek ci joms yra di des nis. Li gos sun ku mas la bai in di vi du a lus, ávai ruo ja pri klau so mai nuo in di vi do am þiaus, et ni - nës gru pës. Be pjautuvinës anemijos, yra ir kitø struktûriniø hemoglobinopati - jø sukeltø ávairiø patologiniø hemoglobinø ar hemoglobino variantø (Hb C, Hb SC, Hb Sd-Los An ge les, Hb SO-Arab ir kt), ta èiau jie yra re tes - ni ar ba ne su ke lia kli ni ki niø simp to mø, to dël ne bus ið sa miai ap ta ria mi. Nors Lie tu vo je ði li ga re ta ir at veþ ti në, bet dël þmo niø mig ra ci jos ið di - de lio pa pli ti mo vie tø he mog lo bi no su tri ki mø bus nu sta to ma vis daþ niau tose ðalyse, kur anksèiau jø nebuvæ. Straips ny je ap þvel gia ma he mog lo bi no struk tû ra, pa pli ti mas, la bo - ra to ri nës diag nos ti kos me to dai, tai ko mi San ta rið kiø kli ni kø la bo ra to ri - nës diag nos ti kos cen tre, taip pat di de lio ðios li gos pa pli ti mo vie to se nau - do ja mi ty ri mo me to dai. Pa grin di niai la bo ra to ri niai ra di niai yra ðie: ávai - raus sun ku mo ane mi ja, re ti ku lo ci to zë, Ho well-jol ly kû ne liai ir làs te lës tai ki niai, ro dan tys hi posp le niz mà ar ba bluþ nies in fark tà, HbS, ku ris pa - tvir ti na mas at lie kant he mog lo bi no elek tro fo re zæ, tei gia mas he mo glo bi - no tir pu mo tes tas. Reikð mi niai þo dþiai: ane mi ja, he mog lo bi nas S, pjau tu vo for mos erit - ro ci tai, he mo li zë, va zo ok liu zi ja, la bo ra to ri në diag nos ti ka. ÁVADAS Pjau tu vi në ane mi ja pri ski ria ma pa - vel di mø struk tû ri niø he mog lo bi no - pa ti jø gru pei. He mog lo bi no pa ti jos tai su tri ki mai, kai pa kin ta he mog lo - bi no struk tû ra, funk ci ja ar ba su trin - ka he mog lo bi no ga my ba. Dël struk tû - ri niø po ky èiø glo bi no po li pep ti di në je gran di në je ga mi na mi pa to lo gi niai he - mo glo bi nai ar ba he mog lo bi no va rian - tai (pa vyz dþiui, HbS, HbC, HbD, HbE ir kt.). Ðios bûk lës daþ niau siai yra ágim tos, jø sun ku mas ávai ruo ja nuo be simp to miø, nu sta to mø tik la bo ra - to rið kai, iki vai siaus þû ties gim do je [1]. Nenormalaus hemoglobino specifinio defekto paieðkos buvo vaisingiausios 1956-ai siais, kai V. M. In gram tiksliai nustatë hemoglobino S amino - rûgð èiø se kà [2]. Ðiuo me tu jau þi no ma daugiau nei 750 struktûriðkai patolo - gi niø he mog lo bi nø. LABORATORINË MEDICINA 2009, t. 11, Nr. 1(41) 21
2 Rëda Matuzevièienë, Asta Maèionienë HEMOGLOBINO STRUKTÛRA Emb rio ni niu, vai siaus lai ko tar piu, taip pat suaugusiø þmoniø kraujyje randami skirtingi hemoglobinai. He - moglobinas tai tetrameras, sudary - tas ið globino polipeptidiniø grandi - niø: po ros ti po gran di niø, su da ry tø ið 141 ami no rûgð ties, ir po ros ti po gran di niø (su da ry tø ið 146 ami no - rûgð èiø). Þmo gaus he mog lo bi nai yra ko duo - ja mi dvie jø ge nø gru piø; tipo globino genai yra 16-oje chro mo so mo je, o ti - po glo bi no ge nai 11-oje chro mo so mo - je. Pa grin di nio nor ma laus su au gu siø - jø he mog lo bi no, HbA, struk tû ra yra 2 2. HbF ( 2 2) vyrauja gestaciniu laikotarpiu, o HbA2 ( 2 2) sudaro ne - didelæ dalá suaugusiøjø hemoglobino. Kiek vie na glo bi no gran di në ap su - pa vienà hemo pusæ, susidedanèià ið proto por fi ri no IX þie do kar tu su vie nu dviva len tës ge le þies (Fe 2+ ) an glies ato - mu taip, kad de guo nis ga lë tø gráþ ta - mai pri si jung ti. Kiek vie na he mo pu së gali prijungti vienà deguonies moleku - læ. Tai gi, kiek vie na he mog lo bi no mo le - kulë gali perneðti iki keturiø deguo - nies molekuliø. Skir tin gø glo bi nø ami no rûgð èiø se kos yra la bai pa na ðios vie na á ki tà. Kiek vie na tu ri ant ri næ spi ra li næ struk tû rà. Dël tre ti nës glo bu li nës struktûros polinës (hidrofilinës) aminorûgðtys iðoriniame pavirðiuje padi - di na tir pu mà, o á vi dø nu kreip tos ne - po li nës gru pës su for muo ja hid ro fo bi næ ki ðe næ, ku rios vi du je yra he mas. Ket - virtinæ HbA struktûrà sudaro di - merai. ir grandines jungia daug tvirtø jungèiø. Visà tetramerà palaiko jung tys tarp vie no di me ro ti po gran - dinës ir ki to di me ro ne ti po gran di - nës. Tirpumas ir gráþtamas deguonies prijungimas dvi esminës hemoglobi - no savybës, kurios gali bûti sutrikdytos sergant hemoglobinopatija. Jos abi daugiausia priklauso nuo aminorûgðèiø hidrofilinio pavirðiaus. Hidrofobinës aminorûgðtys juosia hemo kiðenæ, dau giau sia his ti di nà F spi ra lë je, ir aminorûgðtis, formuojanèias 1 1 ir 2 2 jungtis. Mutacijos ðiuose balty - mo re gio nuo se le mia kli ni ki næ li gos raiðkà. APIBRËÞIMAS Pjautuvinë anemija autosominiu re - cesyviniu bûdu paveldima liga, kurià sukelia taðkinë mutacija globino ge - ne, kuris yra 11p15.4 chromosomoje. Ðio je vie to je ðeð to ji ami no rûgð tis glu - taminas yra pakeièiamas valinu [1]. Ði geno mutacija lemia struktûriðkai ne - nor ma laus HbS ga my bà. HbS yra pir - ma sis pa to lo gi nio he mog lo bi no va - riantas, kuriam nustatyta aminorûgðèiø se ka [2]. PAPLITIMAS PSO duo me ni mis, apie 5 % pa sau lio populiacijos yra ávairiø ágimtø hemoglo bi no va rian tø ne ðio to jai, kas met gims ta apie ávai riai pa þeis tø ho mo zi go tø ar ba he te ro zi go tø [2]. Pjautuvinë anemija daþniausia pa - veldima kraujo liga JAV, kuria serga ame ri kie èiø ar ba 1 ið 500 Af ri - kos amerikieèiø. Ði liga yra didelio ser - gamumo ir mirtingumo Afrikoje prie - þastis, taip pat kiekvienoje populiaci - joje, kur migruoja Afrikos gyventojai, Vidurþemio jûros regione, Viduriniuo - se Ry tuo se, Indijoje [2]. Be homozigotinës HbSS (pjautuvinës ane mi jos), kai 100 % yra ga mi na - mas HbS, þi no mi ne ma þiau kaip pen - ki kiti ligà sukeliantys genotipai: HbS-beta-0 talasemija sunki, fenotipiðkai beveik neatskiriama nuo pjautuvinës anemijos. Pacientas yra dvigubas heterozigotas HbS ir beta-0 talasemijai; HbSC liga vidutinio sunkumo klinika. Pacientas yra dvigubas heterozigotas HbS ir HbC; HbS/paveldima fetalinio hemoglobino (HbF) persistencija lengva ar - ba besimptomë forma; HbS/HbE sin dro mas re ta ir leng va for ma; Re ti de ri niai: HbS kar tu su HbD Los An ge les, HbO Arab, G-Phi l a del p - hia ir kt. Nors Lie tu vo je ði li ga re ta ir at - veþ ti në, bet dël þmoniø migracijos ið di de lio pa pli ti mo vie tø he mog lo bi no sutrikimø bus nustatoma vis daþniau tose ðalyse, kur anksèiau jø nebuvæ. PATOGENEZË Li gos pa to ge ne zæ le mia pa si kei tu si erit ro ci tø struk tû ra ir funk ci ja. Kaip jau minëta, hemoglobinas S atsiranda dël vie nos ami no rûgð ties pa kei ti mo kita (glutamino valinu), dël ðios tað - kinës mutacijos beta globino gene su - sidaro alpha2/betas2 hemoglobino tet - ra me ras. He mog lo bi nas S, ati da væs au di niams de guo ná, tam pa de ok si- HbS, vyksta deoksi-hbs polimerizaci - ja (1 pav.) [3]. HbS po li me rai pluoð tais nu së da ir deformuoja eritrocitus, sutei kia jiems pjau tu vo for mà. Ðie ri gi - dið ki, de for muo ti erit ro ci tai yra ne - lanks tûs, to dël ákliû na smul kio se kraujagyslëse ir jas uþkemða. Tai su - kelia audiniø iðemijà ar infarktà. Pa grin di niai pjau tu vi niø làs te liø ligos patologiniai procesai yra hemoli - zinë anemija ir kraujagysliø uþsikim - ðimas (vazookliuzija) [2]. Vienas ið me - chanizmø, lemianèiø eritrocitø ástrigi - mà mikrocirkuliacijoje, yra sumaþëjæs jø lankstumas. Ástrigæ eritrocitai yra suardomi, dël to sumaþëja jø gyvavi - mo truk më. Taip pat pjau tu vi nës làs - telës yra linkusios prilipti prie endote - lio làsteliø. In vi tro tyrimai rodo, kad tokias làsteles aktyviai atpaþásta mo - nocitai ir makrofagai, todël tikëtina, kad vienas ið anemijà lemianèiø veiksniø yra eritrofagocitozë. Taip pat ima veik ti ir ok si da ci niai me cha niz mai. Pjautuviniø làsteliø membranose yra daug su membrana susijusio hemoch - romo, kuris sukelia membranos kom - ponentø autooksidacijà. Pjau tu vi në ane mi ja pa si reið kia skausmo epizodais (krizëmis) dël ávai - riø organø kraujagysliø okliuzijos, lë - tine hemolizine anemija ir sunkiomis (ypaè in kap su liuo tø mik ro or ga niz mø, pvz., pneu mo ko kø) in fek ci jo mis, kar tu esant trom bo ziø su kel tai asp le ni jai. Heterozigotinë Hb S forma paprastai niekuo nepasireiðkia, iðskyrus ðlapi - mo koncentracijos sutrikimà izoste - nu ri jà. LA BO RA TO RI NË DIAG NOS TI KA La bo ra to ri nës diag nos ti kos me to das parenkamas pagal ligonio amþiø. Bû - tø galima iðskirti prenatalinës diag - nos ti kos me to dus, nau ja gi miø ty ri - mus ir paaugliø bei suaugusiøjø tyri - mus [4]. At ran ki në pre na ta li në diag - nostika, naujagimiø atrankiniai tyri - mai tu ri pras mæ tik di de lio li gos pa pli - ti mo ge og ra fi në se zo no se, to dël pir - miausia apþvelgsime suaugusiø þmo - 1 pav. He mog lo bi no po li me ri za ci ja. Virðuje hemoglobinas esant didelei de - guonies koncentracijai, apaèioje de - oksigenacijos sàlygomis Fig. 1. HbA and HbS un der con di tions of high (abo ve) and low (be low) ox y - gen concentrations 22 LABORATORINË MEDICINA 2009, t. 11, Nr. 1(41)
3 Pjautuvinës anemijos laboratorinë diagnostika 2 pav. Paciento, serganèio hemoglobinopatija S, kraujo tyrimas automatiniu hematologiniu analizatoriumi Fig. 2. Abnormalities of the complete blood cell count of the patient with HbS disease 3 pav. Paciento, serganèio hemoglobinopatija S, morfologinis kraujo tyrimas Fig. 3. Blood cell microscopy results of the petient with HbS disease LABORATORINË MEDICINA 2009, t. 11, Nr. 1(41) 23
4 Rëda Matuzevièienë, Asta Maèionienë 4 pav. VULSK paciento, serganèio hemoglobinopatija S, hemoglobino celiuliozës acetato elektroforezës tyrimas Fig. 4. Cellulose acetate electrophoretogram of the patient with sickle cell ane - mia 5 pav. Pacientø, serganèiø hemoglobinopatija S, hemoglobino tirpumo tyri - mas Fig. 5. Hemoglobin solubility test of the patient with HbS disease niø, ser gan èiø pjau tu vi ne ane mi ja, la - boratorinius tyrimus. Tyrimas pradedamas nuo hemato - loginio ir mikroskopinio kraujo tyri - mo. Su hemoglobinopatija S gali bûti su si jæ to kie au to ma ti nio he ma to lo gi - nio kraujo tyrimo pokyèiai (2 pav.): ane mi ja, re ti ku lo ci to zë, leu ko ci to zë, neut ro fi li ja, trom bo ci to zë, pa di dë jæs erit ro ci tø pa si skirs ty mo plo tas (RDW). Mor fo lo gi niu krau jo ty ri mu (3 pav.) aptinkami ðie pokyèiai: pjau - tuvinës làstelës (pjautuvo formos erit - rocitai), polichromatofilija (dël retikulocitozës), Howell Jolly kûneliai ir làstelës taikiniai, rodantys hipospleniz - mà ar bluþnies infarktà, gigantiniai trombocitai. He mo li zæ ro do kai ku rie bio che mi - niai tyrimai: hiperbilirubinemija (dël ne kon ju guo to he mog lo bi no), LDH (lak tat de hid ro ge na zës) kie kio pa di dë - jimas serume, haptoglobino sumaþëji - mas. Taip pat galimi kiti pokyèiai, ro - dantys organø veiklos nepakankamumà: serumo aminotransferaziø akty - vumo padidëjimas, ðarminës fosfata - zës, serumo kreatinino padaugëjimas. Áta rus he mog lo bi no pa ti jà, rei ka - lingi diagnozæ patvirtinantys labora - toriniai tyrimai: 1. He mog lo bi no elek tro fo re zë ðar - miniame (ph 8.4) celiuliozës acetato ge ly je. 2. He mog lo bi no elek tro fo re zë rûgð - tiniame (ph 6.2) citrato agaro gelyje. 3. He mog lo bi no tir pu mo ty ri mas. 4. He mog lo bi no plo nas luoks në izo - e lek tri nio fo ku sa vi mo elek tro fo re zë ge ly je. 5. He mog lo bi no efektyvioji skys - èiø chro ma tografija (angl. HPLC High per for mance liq uid chro ma tog - ra phy). 6. He mog lo bi no ge nø se kos ty ri - mai. Ce liu lio zës ace ta to elek tro fo re zë ðarminiame (ph 8,4) gelyje yra stan - dar ti nis metodas, kuriuo hemoglobinas S atskiriamas nuo kitø hemoglobi - no va rian tø. Ðar mi nia me tir pa le he - mog lo bi no molekulës ágauna neigiamà krûvá ir elektriniame lauke juda link ano do. Ta èiau HbS, HbG ir HbD elek - tro fo re zi nis jud ru mas su tam pa. No - rint at skir ti HbS nuo HbD ir HbG, rei - kia pa pil do mai at lik ti cit ra to aga ro elek tro fo rezæ rûgðtiniame (ph 6,2) ge - lyje. Ðie metodai turi daugiau istorinæ ver tæ. He moglobino tirpumo tyrimas (pa - vyzdþiui, Sic kle dex) taip pat pa dë tø at skir ti HbD ir HbG nuo HbS, nes tik HbS precipituotø atliekant ðá tyrimà. Tyrimo metu ardomi eritrocitai. Atsipalaidavæs hemoglobinas S yra netirpus koncentruotame 2,3 M fosfatiniame buferiame tirpale. Deoksihemoglobino S polimerai nepraleidþia ðviesos, tir pa las tam pa drums tas (5 pav.). Ðis tyrimas yra naudingas atrankai, kai þmonës tiriami didelio ðios ligos papli - timo vietose. Tyrimas gali bûti klai - din gai tei gia mas, kai yra daug Hein zo kû ne liø, esant ne sta bi liam he mog lo bi - nui po sple nek to mi jos, taip pat dël plazmos baltymø precipitacijos. Klai - dingai neigiamas tyrimas gali bûti dël labai maþo hemoglobino S kiekio sergant sunkia anemija [5]. IZOELEKTRINIO FOKUSAVIMO ELEKTROFOREZË Izo e lek tri nis fo ku sa vi mas yra su dë - tingesnë elektroforezës forma, kai suku ria mas ph gra dien tas (ph nuo 3 iki 6 pav. Pacientø, serganèiø hemoglobinopatija S, hemoglobino izoelektrinio foku - savimo elektroforezës tyrimas Fig. 6. Isoelectric focusing electrophoresis of the patient with hemoglobin S disea se 24 LABORATORINË MEDICINA 2009, t. 11, Nr. 1(41)
5 Pjautuvinës anemijos laboratorinë diagnostika Summary LABORATORY DIAGNOSIS OF SICKLE CELL ANE MIA Rëda Matuzevièienë, Asta Maèionienë 7 pav. Pacientø, serganèiø hemoglobinopatija S, hemoglobino efektyviosios skysèiø chromatografijos tyrimas Fig. 7. HPLC (angl. High per for mance liq uid chro ma tog ra phy) of the pa tient with HbS di s e a se 10). Hemoglobinas, kaip ir kiti balty - mai, yra amfoteriðkas ir gali ágauti tei - giamà arba neigiamà krûvá (priklau - so mai nuo ph). Esant tam tik ram ph, baltymas neteks krûvio ir sustos gelyje tam tik ro je elek tros lau ko vie to je. Ta vieta ir vadinama baltymo izoelek - tri niu tað ku. Izo e lek tri nis fo ku sa vi - mas naudingas naujagimiø tyrimams, kai hemoglobino kiekis yra maþas. HPLC (angl. High per for mance liquid chro ma tog ra phy) me to do ski - riamoji geba yra panaði á izoelektrinio fokusavimo, tik tyrimas yra daug grei - LITERATÛRA 1. Kasper, Braunwald, Fauci, Hauser, Longo. Jameson Har ri son s prin ci ples of internal medicine. 16 th ed. McGraw- Hill, Scriver CR, Beaudet AL, Sly WS, Valle D, Childs B, Kinzler KW, et al. The met a bolic and mo lec u lar bases of in her ited dis ease. 7 th ed. McGraw-Hill; p , 3430, Balsys J. Anemijos. Vilnius: VU lei dyk - la, Vichinsky E, Mahoney D, Gar cia- Prats J, Schrier S, Landaw S, Kim M. tesnis ir patogesnis, labiau tinka at - rankinei diagnostikai (7 pav.). DNR ANA LI ZË He mog lo bino genø sekoskaita (angl. se quenc ing) leidþia identifikuoti tikslià globino geno mutacijà. Analizæ gali - ma atlikti naudojant tiesioginæ sekos kaità arba naudojant aleliui specifi - nius oli go nuk le o ti dus. Gauta: Priimta spaudai: Di ag no sis of sickle cell syn dromes. Prieiga per internetà: < demo/index.html>. 5. McPherson RA, Pincus MR. Henry s clinical diagnosis and management by lab o ra tory meth ods. 21 st ed. Saunders, Burtis C, Ash wood E. Tietz fun da men - tals of clin i cal chem is try. 5 th ed. W. B. Saunders com pany, Sickle-cell ane mia, also called sicklecell dis ease, is a he red i tary dis or der in which ab nor mal he mo glo bin (HbS) within the red blood cells causes the cells to take on ab nor mal sickle shapes. He mo glo bin S re sults from the sub - sti tu tion of a valine for glutamic acid of the beta glo bin chain which pro duces a he mo glo bin tetramer that is poorly sol - u ble when deoxygenated. He mo glo bin poly mer iza tion, changes in red cell mem brane struc ture and func tion de - creases the abil ity of the he mo glo bin to transport oxygen throughout the body, causes hemolysis and vasooclusion. Mul ti ple vasooclusion events and chronic ane mia cause car diac, pul mo - nary, gen i to uri nary, sceletal, neu ro - logic, oc u lar and dermatologic dis or - ders. One com pli ca tion is phe nom ena of autosplenectomy. Be cause of this, these pa tients have in creased sus cep ti - bil ity to in fec tions. The se ver ity of the dis ease varies from individual to individual and between ethnic groups. There are var i ous sickling dis or - ders caused by more than 750 struc - tural he mo glo bin vari ants but most of them are rare and of no clin i cal or hematologic sig nif i cance. He mo glo bin struc ture, lab o ra tory di ag no sis of pa tients with sickle cell dis ease will be re viewed here. The main lab o ra tory fea tures of sickle cell ane mia in clude vari able ane mia, reti - cu lo citosis, the pres ence of Howell- Jolly bod ies and tar get cells in di cat ing hyposplenism or splenic in farc tion, HbS on he mo glo bin elec tro pho re sis and a positive solubility test. Keywords: ane mia, he mo glo bin S, sickle cells, hemolysis, vasooclusion, lab o ra tory di ag no sis. LABORATORINË MEDICINA 2009, t. 11, Nr. 1(41) 25
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