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Klini ki zavod za laboratorijsku dijagnostiku, KBC Zagreb, Zagreb Uvod U National Academy of Clinical Biochemistry, National Comprehensive Cancer Network (NACB i NCCN) smjernicama za upotrebu tumorskih biljega u monoklonskim gamapatijama ve se 2006. godine spominje imunotipizacija kao postupak za definiranje razreda i tipa monoklonskog proteina (M-proteina). U istim se smjernicama navodi da se imunotipizacija provodi metodom imunofiksacije ili imunosuptrakcije. Imunosuptrakcija nije nova metoda. Joa su 1977. godine Aguzzi i Poggi opisali imunosuptrakciju kao metodu koja omoguava precipitaciju specifi nih proteina upotrebom monospecifi nog antiseruma prije izvoenja elektroforeze na acetat celulozi. Uo ili su da uklanjanje specifi nih proteina (imunosuptrakcija) prije elektroforeze omoguava bolju interpretaciju krajnjeg elferograma.(1) Za detekciju M-proteina prvi su imunosuptrakciju upotrijebili White i Attwood, a smjernice za klini ku i laboratorijsku evaluaciju bolesnika s monoklonskim gamapatijama iz 1999. zavraavaju opisujui imunosuptrakciju kao obeavajuu metodu za tipiziranje M-proteina.(2) Imunotipizaciju treba provesti u slu aju postojanja izra~ene homogene vrpce odnosno vraka u podru ju od alfa2 do gama globulina u elferogramu, kod postojanja klini ke sumnje na monoklonsku gamapatiju iako ne postoji izra~en vraak u elferogramu te kod pacijenata kod kojih se M-protein prvi puta izgubio kao posljedica uspjeane terapije. Posljednje se desetljee zbog mogunosti automatizacije kapilarna zonska imunosuptrakcija sve viae primjenjuje u klini kim laboratorijima s velikim brojem uzoraka. Imunofiksacija Imunofiksacija je postupak koji predstavlja  zlatni standard u definiranju razreda i tipa M-proteina. Danas je to poluautomatizirani postupak koji uklju uje elektroforezu proteina na agaroznom gelu, imunoprecipitaciju i vizualizaciju nespecifi nim bojenjem (slika1.). Nakon elektroforeze proteina istog seruma u naj eae 5 linija svaka se linija na gelu inkubira sa specifi nim antiserumom za odreeni razred teakog lanca (anti , anti , anti ) i tip lakog lanca (anti 8, anti ). Antiserum reagira s odgovarajuom imunoglobulinskom domenom tvorei kompleks koji ostaje fiksiran u gelu. Ostali nevezani proteini se ispiru s gela. Nakon nespecifi nog bojenja proteina vidljiv ostaje samo fiksirani protein. Monoklonski protein se pojavljuje kao ja e izra~ena vrpca na slabije obojenoj pozadini koja predstavlja zadr~anu poliklonsku sintezu imunoglobulina istog razreda.(3) Ako postoji sumnja na prisutnost M-proteina, a nije uo ena reakcija s anti-IgA, anti-IgM i anti-IgG antiserumima preporu a se uzorak testirati anti-IgD i/ili anti-IgE antiserumima.  Slika 1. Shematski prikaz postupka imunofiksacije Imunosuptrakcija Za razliku od imunofiksacije, imunosuptrakcija je danas potpuno automatizirani postupak koji se sastoji od imunoprecipitacije, elektroforeze proteina kapilarnom zonskom elektroforezom i UV detekcije razdvojenih proteina (slika 2.). U ja~icama segmenta na nosa u uzorka nalazi se diluent i kuglice sefaroze oblo~ene odgovarajuim antiserumom (anti , anti , anti , anti 8, anti ). Neposredno prije elektroforeze alikvoti seruma se inkubiraju s oblo~enim kuglicama sefaroze. Nastali kompleksi zaostaju u otopini, a u 6 silikoniziranih kapilara istovremeno se pod istim uvjetima provodi elektroforeza proteina. Svaka elektroforeza uzorka seruma inkubiranog sa kuglicama sefaroze usporeuje se pomou ra unalnog programa s elektroforezom nativnog uzorka seruma.  Slika 2. Shematski prikaz postupka imunosuptrakcije Tuma enje nalaza imunosuptrakcije Nestanak ili promjena simetri nosti odgovarajue frakcije u elferogramu uzorka inkubiranog s kuglicama sefaroze oblo~enim odgovarajuim antiserumom ukazuje na nastanak kompleksa sa specifi nim antiserumom, odnosno ukazuje na razred i tip prisutnog monoklonskog proteina (slika 3.). Treba istaknuti da e do reakcije imunoprecipitacije tj. stvaranja kompleksa doi ne samo i isklju ivo u prisutnosti M-proteina, ve e odgovarajui antiserum stvoriti kompleks i s odgovarajuim poliklonski sintetiziranim imunoglobulinom. Upravo zbog toga interpretacija rezultata imunosuptrakcije iziskuje educirano i iskusno stru no osoblje.(3) Naime, kako 80% imunoglobulina ini IgG najvei pad simetri ne zvonolike krivulje imunosuptrakcije u uzorku s normalnom poliklonskom sintezom imunoglobulina dogoditi e se u podru ju gama frakcije. Manji pad krivulje u podru ju beta frakcije odgovarati e imunoglobulinu A koji ini 15% imunoglobulina. Prisutnost IgM (5%) o itovat e se u neznatnom padu krivulje u podru ju gama globulina. Budui da je fizioloaki omjer kapa i lamda lanaca 2:1, pad krivulje u uzorku obraenom s anti-kapa antiserumom biti e dva puta vei od onoga s anti-lambda antiserumom (slika 4.). Na prisutnost M-proteina treba pomisliti pri uo avanju neuobi ajene frakcije u podru ju od alfa2 do gama globulina, prilikom porasta beta frakcije bez porasta alfa 1 i alfa2 globulina, u slu aju da je frakcija beta2 vea od frakcije beta1 te u slu aju gubitka simetri nosti bilo koje frakcije u elferogramu. U pravilu se relativno lako tipiziraju M-proteini koncentracije vee od 10g/L. Kada koncentracija M-proteina padne ispod 5g/L sve je te~e interpretirati rezultate imunosuptrakcije. Stoga se preporu a slo~enije elferograme, hipogamaglobulinemiju, biklonalne gamapatije, moguu prisutnost monoklonskih slobodnih lanaca tipizirati/potvrditi metodom imunofiksacije. (4,5)  Slika 3. Prikaz rezultata imunosuptrakcije usporedbom referentnog elferograma sa elferogramima uzorka nakon inkubacije sa specifi nim antiserumima. Imunosuptrakcija uzorka s monoklonskim proteinom IgA kapa tipa. Pregledom referentnog elferograma uo ava se izra~en vraak u podru ju beta frakcije. Nakon reakcije s antiserumima na alfa teaki lanac i kapa laki lanac doalo je na istom mjestu do potpunog pada krivulje.  Slika 4. Prikaz rezultata imunosuptrakcije usporedbom referentnog elferograma sa elferogramima uzorka nakon inkubacije sa specifi nim antiserumima. Imunosuptrakcija uzorka s poliklonskom hipergamaglobulinemijom. Pregledom referentnog elferograma uo ava se poveana beta2 frakcija te podru je izmeu beta i gama frakcije.Nakon reakcije s antiserumima doalo je do simetri nog pada krivulje na svim elferogramima. Pad krivulje s anti-kapa antiserumom duplo je vei od pada krivulje s anti-lambda antiserumom ato odgovara fizioloakom omjeru kapa/lambda lanaca. Zaklju ak Tablica 1. Usporedni prikaz karakteristika metoda za imunotipizaciju M-proteina ImunotipizacijaImunofiksacijaImunosuptrakcijaElektroforezaGel elektroforezaKapilarna elektroforezaNosa  elektroforezeAgarozaOtopinaMogunost detekcije M-proteinaIgA, IgG, IgM, IgD, IgEIgA, IgG, IgMAutomatizacijaPoluatomatiziranaPotpuno automatiziranaTrajanje2h10minTehni ka izvedbaPotrebna vjeatina-Interpretacija rezultataPotrebno iskustvoPotrebno iskustvo Literatura  HYPERLINK "http://europepmc.org/search;jsessionid=s2y7ypHwomtrAcogNuYS.0?page=1&query=AUTH:"Litwin+CM""Litwin CM, HYPERLINK "http://europepmc.org/search;jsessionid=s2y7ypHwomtrAcogNuYS.0?page=1&query=AUTH:"Anderson+SK""Anderson SK, HYPERLINK "http://europepmc.org/search;jsessionid=s2y7ypHwomtrAcogNuYS.0?page=1&query=AUTH:"Philipps+G""Philipps G, HYPERLINK "http://europepmc.org/search;jsessionid=s2y7ypHwomtrAcogNuYS.0?page=1&query=AUTH:"Martins+TB""Martins TB, HYPERLINK "http://europepmc.org/search;jsessionid=s2y7ypHwomtrAcogNuYS.0?page=1&query=AUTH:"Jaskowski+TD""Jaskowski TD, HYPERLINK "http://europepmc.org/search;jsessionid=s2y7ypHwomtrAcogNuYS.0?page=1&query=AUTH:"Hill+HR""Hill HR. Comparison of capillary zone and immunosubtraction with agarose gel and immunofixation electrophoresis for detecting and identifying monoclonal gammopathies. Am J of Clin Pathol1999;112:411-417 David F. Keren, Raymond Alexanian, James A. Goeken, Peter D. Gorevic, Robert A. Kyle, Russell H. Tomar. Guidelines for Clinical and Laboratory Evaluation of Patients With Monoclonal Gammopathies. Arch Pathol Lab Med.1999;123:106 107 David F Keren. Protein Electrophoresis in Clinical Diagnosis, CRC Press 2003. Christopher R. McCudden, Stephanie P. Mathews, Shirley A. Hainsworth, John F. Chapman, Catherine A. Hammett-Stabler, Monte S. Willis, David G. Grenache. Performance Comparison of Capillary and Agarose Gel Electrophoresis for the Identification and Characterization of Monoclonal Immunoglobulins. Am J Clin Pathol 2008;129:451-458 Zhaohai Yang, Keith Harrison, Yara A. Park, Carolyn H. Chaffin, Beatrice Thigpen, Pattye L. Easley, i sur. Performance of the Sebia CAPILLARYS 2 for Detection and Immunotyping of Serum Monoclonal Paraproteins. 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