Nora Nikolac. Klinički zavod za kemiju, KBC Sestre milosrdnice
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1 Lipemija Nora Nikolac Klinički zavod za kemiju, KBC Sestre milosrdnice
2 Lipemija Zamućenje seruma ili plazme uzrokovano povišenom koncentracijom lipoproteina Učestalost: < 1% uzoraka 10-15% nesukladnih uzoraka 2
3 Lipoproteini Čestice koje pridonose turbiditetu uzorka Hilomikroni VLDL LDL HD L nm 6-12,5 nm nm velike srednje male nm nm nm 3
4 Uzroci lipemije Predanalitički čimbenici Patofiziološka stanja akutni pankreatitis, alkoholizam, kronično zatajenje bubrega, hipotireoidizam, multipli mijelom, primarna bilijarna ciroza, sistemski lupus, inzulinska rezistencija, šećerna bolest, lijekovi 4
5 Mehanizmi djelovanja lipemije Interferencije zbog fizikalno-kemijskih čimbenika Interferencije u spektrofotometrijskim metodama Nehomogenost uzorka Efekt promjene volumena 5
6 Interferencije zbog fizikalno-kemijskih čimbenika Blokiranje Ag / At u imunokemijskim metodama Učinak matriksa na kromatografske i elektroforetske tehnike 5,2 mmol/l TG 3,1 mmol/l TG 2,1 mmol/l TG 0,5 mmol/l TG Dodatak uzorka sa rastućom konc. TG rezultira abnormalnom morfologijom u zoni alfa-2-globulina. Bossuyt X, et al. Serum protein electrophoresis by CZE 2000 clinical capillary electrophoresis system. Clin Chem 1998;44:
7 Interferencije u spektrofotometrijskim nefelometrija metodama Detektor 90 -Veličina čestica -Broj čestica Izvor svjetla turbidimetrija Detektor 180 spektrofotometrija 7
8 Nehomogenost uzorka Hidrofobna (lipidna) faza Lažno hidrofilni analiti Hidrofilna (vodena) faza Lažno hidrofobni analiti 8
9 Efekt promjene volumena Smanjenje vodene faze u lipemičnim uzorcima Analiti koji se raspodjeljuju u vodenoj fazi, a ne mjeri im se aktivnost Određivanje koncentracije elektrolita plamena fotometrija indirektna potenciometrija direktna potenciometrija razrjeđenje uzorka konc. u ukupnom volumenu plazme bez razrjeđenja konc. u vodenoj fazi 9
10 Efekt promjene volumena Normalan uzorak K + Na + Cl - K + Na + Cl- Lipemija 92% vode Plamena fotometrija Indirektna potenciometrija Na + = K + Na + K + Cl- Cl - Na + 8 % lipida 145 mmol/l = 145 mmol/l 145 mmol/l Direktna potenciometrija Na + = = 158 mmol/l 0,92 L 1 L K + Na + Cl - 25 % lipida Na + K + Cl mmol/l 1 L 118 mmol/l 0,75 L 75% vode = 118 mmol/l = 158 mmol/l 10
11 Metode identifikacije lipemije Vizualna identifikacija Određivanje koncentracije triglicerida Analitički sustavi 11
12 Vizualna identifikacija lipemije Serum/plazma TG > 3,4mmol/L Puna krv TG >11,3mmol/L Ovisno o vrsti lipoproteina Nativna lipemija TG (mmol/l) 1,25 1,75 2,22 3,09 5,06 8,85 Lipemija simulirana dodatkom Intralipida 12
13 Određivanje koncentracije triglicerida Različit udio TG u pojedinim lipoproteinima Slaba korelacija TG i indeksa lipemije za nativno lipemične uzorke Twomey PJ, et al. Unreliability of triglyceride measurement to predict turbidity induced interference. J Clin Pathol 2003;56:
14 Određivanje koncentracije triglicerida Lažno konc. uz glicerol TG = 27,5 mmol/l, lipemijski indeks, osmolarni gap 14
15 Analitički sustavi Intralaboratorijska standardizacija procjene Interlaboratorijska varijabilnost Mjera raspršenja svjetlosti na velikim česticama Razlike u apsorbanciji na različitim valnim duljinama: 660/700, 550/524, 572/604, 524/804 nm Lažno pozitivni rezultati 15
16 Analitički sustavi IgM = 26 g/l (0,4-2,3) IgG = 4,6 g/l (7,0-16,0) IgA = 0,62 g/l (0,70-4,0) Κ = 1,40 g/l (0,38-3,75) λ = 3,18 (0,93-2,42) κ/λ = 0,44 (1,17-2,93) L indeks = 4000 (< 1000) bistar uzorak Potrebna naknadna vizualna provjera uzoraka sa visokim indeksima. 16
17 Metode uklanjanja lipemije Priprema bolesnika Razrjeđenje uzorka Korekcija rezultata Precipitacija lipoproteina Centrifugiranje Ekstrakcija lipofilnim otapalima 17
18 Centrifugiranje Zlatni standard za uklanjanje lipemije Vrsta centrifuge Ultracentrifuga ( , g) Centrifuga s visokim brojem okretaja ( g ) Centrifuga ( g) 18 Dimeski G, Jones BW. Lipaemic samples: Effective process for lipid reduction using high speed centrifugation compared with ultracentrifugation. Biochem Med 2011;21:86-94.
19 Centrifugiranje Učinkovitost centrifugiranja ovisi o veličini čestica Hilomikroni se učinkovitije uklanjaju U bistrom sloju nema analita koji se raspodjeljuju u mastima (oprez: lijekovi, hormoni) 19
20 Ekstrakcija lipofilnim otapalima Ekstrakcija lipoproteina u lipofilnu fazu Netoksični neionski polimer Lipoclear(StatSpin, Norwood, MA, SAD) 20
21 Lipoclear Usporedba učinkovitosti delipidiranja reagensom Lipoclear u odnosu na ultracentrifugiranje Lipemija izazvana Intralipidom Vermeer HJ, Steen G, Naus AJ, Goevaerts B, Agricola PT, Schoenmakers CH. Correction of patient results for Beckman Coulter LX-20 assays affected by interference due to hemoglobin, bilirubin or lipids: a practical approach. Clin Chem Lab Med 2007;45:
22 Lipoclear 22
23 Problemi ispitivanja interferencija Nativna lipemija Lipemija simulirana dodatkom Intralipida Intralipid: sojino ulje, fosfolipidi iz žumanjka jajeta i glicerin 20%-tna otopina bogata trigliceridima oleinske, linoleinske, palmitinske i stearinske kiseline 23
24 Intralipid Veličina čestica: 345 nm ( nm) Čestice koje pridonose turbiditetu uzorka Hilomikroni VLDL LDL HDL I I nm 6-12,5 nm nm velike srednje male nm nm nm 24
25 Upute proizvođača Proizvođač, test Beckman Coulter, Haptoglobin Roche, Prokalcitonin Deklaracija <5% pri 1000 mg/dl Intralipida ± 15% do 1500 mg/dl Intralipida Abbott, Valproična kiselina Siemens, Alfa-1-antitripsin Ortho-Clinical Diagnostic, Etanol < 10% pri 1100 mg/dl triglicerida Nema interferencije do 8,2 g/l triglicerida < 11 mg/dl pri 1600 mg/dl triglicerida Prikaz interferencije zbog lipemije nije standardiziran 25
26 Učinak Intralipida Bornhorst JA, Roberts RF, Roberts WL. Assay-specific differences in lipemic interference in native and intralipid-supplemented samples. Clin Chem 2004;50: Ultracentrifugiranje Koncentracije proteina: AAT, CER, HPT, PRE, TRF Iskorištenje Interferencije Koncentracije proteina: AAT, CER, HPT, PRE, TRF Lipemija simulirana Intralipidom Nativno lipemični uzorci Lipemijski indeks Koncentracije proteina: AAT, CER, HPT, PRE, TRF Intralipid do identičnog lipemijskog indeksa 26
27 Učinak Intralipida Nativno lipemični uzorci Ultracentrifugiranje Lipemija simulirana Intralipidom CER 52% PRE 38% TRF 14% AAT - HPT - CER 7% PRE - TRF - AAT - HPT - Nativna lipemija nije identična lipemiji simuliranoj Intralipidom Oprez pri interpretaciji uputstava proizvođača! 27
28 Završno Pisani protokol za postupanje s lipemičnim uzorcima Provjerapreporuka proizvođača o utjecaju lipemije Lipemiju uzorka naznačiti na nalazu Na koje pretrage utječe lipemija Odabir protokola delipidiranja prema vrsti traženih analita 28
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