Marilyn Johnston Hemostasis Reference Laboratory Hamilton,Ontario

Transcription

1 Marilyn Johnston Hemostasis Reference Laboratory Hamilton,Ontario

2 None

3 Discovered in 1916 Named from the Latin word for liver, hepar 1939, heparin requires a plasma cofactor First used in humans in , cofactor identified as AT 1979, mechanism of Hep-AT interaction 1980 s LMWH introduced

4 XIIa XIa VIII VIIa IXa ATIII/Heparin V Va Fibrinogen Xa IIa Fibrin Figure 17.1 Heparin/antithrombin (AT) irreversibly inhibits the coagulation enzymes: Thrombin and factor Xa are most sensitive to the effects of heparin/at. Adapted from Hirsh,

5 Anionic mucopolysacchride - found in mast cells - isolated from mucosa MW, 3,000 30,000 daltons Plasma half life - 60 minutes

6 Prepared from UFH *enzymatic *chemical Half life: ~ 2hours MW to 8000 Daltons

7 UFH Heparin Molecular Weight 16,000 DEPOLYMERIZATION LMWH Molecular Weight 4,000-5,000 High Affinity for ATIII Figure 17.2 Commercial-grade heparin is chemically or enzymatically depolymerized to create low molecular weight heparin which contains the high-affinity pentasaccharide. Adapted from Hirsh,

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9 Pharmacokinetic Bioavailability not predictable nonspecific binding to proteins and cells Biophysical failure to inactivate thrombin bound to fibrin Biological HIT and Osteopenia

10 Can be dosed in a predictable fashion -limited binding to proteins -does not bind to endothelium or macrophages Requires limited or no monitoring Can be used for outpatient treatment

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12 Pentasaccharide-containing fractions with less than 18 saccharide units 50-75% Pentasaccharide-containing fractions with 18 or more saccharide units 25-50% AT Thrombin AT Thrombin 5 LMWH 5 No inhibition of thrombin Inhibition of thrombin LMWH AT Xa AT Xa 5 Inhibition of factor Xa (Xa) LMWH 5 Inhibition of factor Xa LMWH Figure 17.3 Low molecular weight heparin activity. Approximately 25 to 50% of the LMWH molecules of different commercial preparations contain at least 18 saccharide units; these molecules inhibit both thrombin and factor Xa. The remaining 50 to 75% of LMWH molecules contain fewer than 18 saccharide units and only inhibit factor Xa. Adapted from Hirsh,

13 LMWHs Dalteparin (Fragmin) Enoxaparin (Lovenox) Tinzaparin (Innohep) Reviparin (Clivarine) Nadroparin (Fraxiparine) Ardeparin (Normiflo) Anti- Xa to anti- IIa MW range ratio 2.7:1 2,000 9, :1 3,000 8, :1 3,000 6, :1 Mean MW :1 Median MW :1 Mean MW 6000

14 Inexpensive and readily available BUT Therapeutic range is reagent, instrument and lot # dependent Not specific for heparin

15 Prolonged APTT *warfarin therapy *antiphospholipid antibody syndrome * liver disease Short APTT *acute phase reactants -VIII and fibrinogen suggests inadequate response - labeled heparin resistance

16 For UFH

17 Do Not Spike heparin INTO NORMAL POOLED PLASMA To establish a therapeutic range

18 Difference in the MW distribution High MW heparin cleared faster (anti 11a) EX VIVO

19 Collect blood from 30 to 50 patients * intravenous heparin >4-6 hours after bolus >less than 24 hours after 1 st dose of Warfarin Prepare platelet poor plasma Brill Edwards et al Ann Intern Med 1993:119:

20 Measure APTT and heparin level Plot X axis - heparin and Y axis - aptt Perform a regression analysis Determine the aptt range that correlates to u/ml* anti Xa units * * Levine et al, Ann Inter Med 1993;119:

21 Heparin Regression

22 Freeze PPP with known anti- Xa levels Before changing lot numbers run aptt with old and new lot # s If within the tolerance of the assay accept If not; plot results and perform regression analyses and calculate new range or repeat the 1 st protocol

23 Use split samples Measure old and new lot numbers Calculate difference between pairs - 5 sec. difference is acceptable sec. is of concern - >7 sec. action must be taken. Olsen et al; Arch Pathol Lab Med 1198; 122:

24 different reagents Perform a full protocol Set a new therapeutic range and inform the physicians of the change

25 No interference from coagulation pathway Monitor LWMH Automated DISADVANTAGE: $$$

26 Rosborough TK Pharmacotherapy 1999;19: Monitored 268 patients treated with UFH with anti Xa and aptt RESULTS: fewer dose adjustments in the anti Xa group compared to aptt group Fewer tests were required Over a 96 hour period, the Xa assay cost $4.37 more

27 Anti Xa Assays Heparin + AT(plasma and exogenous) >>> [Hep-AT] [Hep-AT] + [FXa (excess)] [FXa-AT-Hep.] + [residual FXa] [residual FXa] + Substrate >>>Peptide + pna

28 Manufacture Concentration Antithrombin Concentration Xa Buffer Substrate Calibrators available Hyphen Biomed Human (~40 ug/ml) Bovine ~6 ug/ml = ~11.4nkats/ml Tris-NaCl- EDTA (ph 7.85) CS 11(65) 2 mg/ml UFH & LMWH Chromogenix Coatest Human (1 IU/ml) Bovine 7.1nKats/ml Tris S mmole/l LMWH Siemans Diagnostics Human (1 IU/ml) Human? Tris-NaCl- EDTA + Dextran sulphate Z-D-leu-glyarg-ANBAmethyl amide 4mmol/L no IL Human (1 IU/ml) Bovine 13.6 nkats/ml Tris- NaCL,EDTA ph8.4 S2765 no Biopool Spectrolyse Heparin Actichrome Heparin (antifxa) Human Bovine 4 nkat/ml 0.05M Tris HCl, 0.175M NaCl, 7.5mM EDTA, ph 8.4 MeO-COD- CHG-Gly- Arg-pNA 4 mmol/l no Stago Bovine? Bovine 4.1 nkats/ml Tris EDTA (ph 8.4) CBS 31: umoles /ml UFH & LMWH (reference WHO Std)

29 . Comparison of commercial one stage anti-xa.heparin assays. Manufacture Concentration Antithrombin Concent. Xa Buffer Substrate STAGO - not used IU/ml Components CBS Rotochrom (5.5 nkat/ml) not given 1umole/ml CHROMOGENIX not used 7 nkat per ml Tris,EDTA. S-2732 Coamatic co- NaCl 3 mg/ml lyophilized dextran with buffer sulfate, BSA

30 Hyphen Biomed IL Liquid heparin assay

31 Reagent with AT supplemented -measures all the heparin present in the plasma Reagent without AT -measures the functional heparin -dependent on patients AT levels

32 UFH standard (WHO 5 th International Std. LMWH standard (WHO 2 nd International Std) Manufactures secondary standards must be referenced against the WHO material Some assays use a hybrid line.? Can 1 LMWH standard be used for all LMWH

33 1200 Optical Density Fraxiparine Tinzaparin Enoxaparin Dalteparin Clivarin Concentration

34 Fragmin Preparations Optical Density WHO Line Dalteparin Injection Stago Line Concentration

35 When different LMWH preparations were compared, a two way ANOVA showed statistically significant differences in slopes and intercepts. When Fraxiparin was removed from the analysis, no differences were seen. The differences seen were not clinically relevant when patient results were compared. The 3 different preparations of Fragmin gave comparable patient results.

36 Commerical controls can be used Two levels are required Suggest making an inhouse control with the same heparin used for treatment

37 Anti 11a Assays Heparin + AT [Hep-AT] [Hep-AT] + [excess IIa] [FIIa-Hep-AT] + [residual IIa] [IIa (residual)] + IIa-Substrate Peptide + pna

38 Careful Venipuncture mol buffered citrate - CTAD tubes (citrate, theophylline,adenosine and dipyridamole) Centrifuge within 1 hour of collection G for 15 min, remove plasma and centrifuge again for 5 min. or - filter using a 0.22 um filter

39 aptt is most common test for UFH Difficult to establish a therapeutic range In vitro spiking of plasma is not recommended aptt cannot be used for monitoring LMWH

40 Heparin assays are specific -not affected by other anticoagulants or factors - anti-xa is the only assay to monitor LMWH -achieve a higher % of therapeutic values within 24 hours Specific LMWH standards appear not to be necessary for calibration.