Heparin time assays

Therapy, in the short term, is with intravenous unfractionated or subcutaneous low molecular weight heparin. Aspirin, given in low doses between 50 and 100 mg per day, is sufficient to diminish platelet-vessel interaction. Alternatives include 100-200 mg of sulphinpyrazone once or twice a day or dipyridamole where 100 mg four times a day can be used on its own or between 25 and 75 mg combined with aspirin three times a day. More recently thiopy-ridines, as a class, has been shown to have equivalence at 250 mg twice a day. In hyperhomocysteinaemia the risk is reduced by 5 mg of folate and 100 mg of vitamin Bg daily, with addition of oral vitamin Bi2 of less clearly defined benefit. The effect of this intervention requires re-assay at 3-monthly intervals, following standard methionine challenge, to ensure that suitable suppression has been achieved in the plasma amino acid level (Table 5). 

For blood heparinized venous blood is centrifuged and the upper layer is removed. Wash the erythrocyte sediment three times with 0.9% (w/v) NaCl solution. Haemolyse the washed red cells by adding 4 parts (v/v) of distilled water per volume of packed cells to give a stock haemolysate solution (approx. 5% (w/v)). For assay, dilute the stock solution 1 500 with the phosphate buffer immediately before the assay is to be carried out and determine the haemoglobin content of the solution by the method of Drabkin. The catalase activity is expressed per unit of haemoglobin. 

Artefactual increases of as much as 50% in total thyroxine, estimated by a competitive protein-binding assay, and of as much as 30% in triiodothyronine resin uptake are probably due to rapid and continuing lipolytic hydrolysis of triglycerides after blood has been drawn (126). Thyroid function tests should therefore always be performed on blood samples taken before (or a sufficient time after) heparin treatment (127). An increase in serum-free thyroxine concentrations has also been reported after low molecular weight heparin, by up to 171% in specimens taken 2-6 hours after injection. When specimens were obtained 10 hours after injection, the effects were smaller, but with concentrations still up to 40% above normal the results can still cause errors of interpretation (128). 

Ionic contrast media canse temporary prolongation of clotting time in patients treated with heparin this effect may last for 6 honrs and may interfere with laboratory assays (132). Non-ionic media do not have this anti-coagnlant effect, and if blood is allowed to mix with a non-ionic medinm in the sjringe or catheter, thrombus formation can occnr, which could be a potential cause of thromboembolism (SEDA-15, 502). 

Sample material Serum or heparin plasma. Use only fresh material. Samples that have been stored for some time are unsuitable for the assay. 

Patients need not be fasting. Venous blood should be collected in tubes containing EDTA, oxalate, or fluoride. Sample stability depends on the assay method. Whole blood may be stored at 4 °C for up to 1 week. Above 4 °C, Hb Ai +b increases in a time- and temperature-dependent manner, but Hb Ale is only slightly affected. Storage of samples at -20 °C is not recommended. For most methods, whole blood samples stored at -70 °C are stable for at least 18 months. Heparinized samples should be assayed within 2 days and may not be suitable for some methods of analysis (e.g., electrophoresis). 

Serum, heparinized plasma, whole blood, sweat, urine, feces, or gastrointestinal fluids may be assayed for Nah Timed collections of urine, feces, or gastrointestinal fluids are preferred to allow comparison of values with reference intervals and to determine rates of electrolyte loss. Serum, plasma, and urine may be stored at 2 C to 4 C or frozen. Erythrocytes contain only one tenth of the Na" present in plasma, so hemolysis does not cause significant errors in serum or plasma Na values. Lipemic samples should be ultracen-trifuged and the infranatant analyzed unless a direct ISE is used. 

Heparin therapy may be monitored by its increases in clotting time in the APTT, although this method for measuring heparin is difficult to standardize. Heparin is more specifically assayed by its effect on factor Xa inactivation by antithrombin. Such factor Xa-based heparin assays usually employ purified factor Xa as a reagent and factor X-deficient substrate plasma as the source of antithrombin. The prolongation of the clotting time that results from the heparin in the patient s plasma is compared with pooled normal plasma that is known to be free of heparin. Many variations of this heparin assay are available. Heparin assays can use thrombin rather than factor Xa, however, the low-molecular-weight heparins are not reliably measured in thrombin-based assays. 

When anti-factor Xa activity is used to monitor LMWH therapy, the sample should be drawn approximately 4 hours after the subcutaneous injection, during the peak period of anti-factor Xa activity. A calibrated LMWH heparin should be used to establish the standard curve for the assay. The therapeutic range for anti-factor Xa activity is not well defined and to date has not been correlated clearly with efficacy or the risk of bleeding. Eor the treatment of VTE, an acceptable target range is 0.5 to 1.0 unit/mL. Specific algorithms for dosing adjustments based on anti-factor Xa activity are not available at the present time. 

The assay used by Pattnaik et al. (1978) to isolate the cholesteryl ester transfer protein in human plasma measured the transfer of 3H-labeled cholesteryl ester from low density to high density lipoprotein. The transfer reaction was terminated by adding MnC and centrifuging to sediment the low density lipoprotein. A heparin, MnCl2 precipitation step may also be used however, in the presence of a phosphate buffer, heparin is not necessary for precipitation of low density lipoprotein. Routine assays were found to be reproducible to within 10%. The increase in high density lipoprotein radioactivity is linear with time until about 25% of the initial low density lipoprotein radioactivity is transferred. The lipoproteins may also be separated by ultracentrifugation. When these two methods were compared, the results agreed within 10%. The precipitation technique is much faster (less than 15 min compared with 18 hr) and simpler. 

A pharmacokinetic comparison of both product generations, and their formulation buffers was conducted in partially heparinized Sprague-Dawley rats using a monoclonal antibody capture/activity assay. The two rFVIII concentrates exhibited a high degree of similarity in half-life, area under the curve (AUC), clearance rate, and mean retention time, predicting biological activity of rAHF-PFM in humans similar to that of rAHF. 

Displacement by plasma of radiolabeled thrombin and radio-labeled thrombin-antithrombin III inactive complex from a heparinized surface was measured and found to be significant for example, removing 63% of the thrombin and 90% of the complex that could not be removed by phosphate-buffered saline alone. Heparin-poly(vinyl alcohol) (PVA) gel beads with a very low heparin release rate, prepared by acetal coupling of the heparin to the PVA, adsorbed thrombin and potentiated the inactivation of thrombin by antithrombin 111, as measured by both thrombin time and chromogenic substrate assays. 

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