Pre-analytical conditions affecting the determination of the plasma homocysteine concentration

In the past decade, moderately elevated homocysteine concentration has achi eved wide-spread recognition as an independent risk factor for vascular dis eases, such as stroke and peripheral vascular disease, as well as for an im paired nutritional status. In general, EDTA plasma is used for the determin ation of homocysteine. However, from the pre-analytical point of view it is important, that, when plasma is not separated from blood cells within 30 m inutes, homocysteine levels increase in samples significantly by about 10% per hour. This 10% increase is very important, because the normal range is between 5 and 15 mu mol/l and moderately elevated homocysteine concentratio ns above 15 mu mol/l may signify an increased risk of vascular disease. The se preliminary cut-off points show that there is only a small difference be tween normal and moderately elevated homocysteine concentrations. Most blood samples are obtained outside the hospital, and in these cases ho mocysteine concentrations will be falsely elevated, if no precautions are t aken, such as immediate centrifugation and separation of plasma and cells. This aspect is critical both for clinical studies and in patient care outsi de the hospital. But even in the hospital it is difficult to separate plasm a and cells within 30 minutes. In the past, different approaches were adopted to solve this problem. Poten tial stabilisers were sodium fluoride (4 g/l) and 3-deazaadenosine (100 mu mol/l). Sodium fluoride initially increased the homocysteine concentration, which dropped below the initial values after 72 h. On the other hand, 3-de azaadenosine stabilised homocysteine concentrations for 24 h, but increased it within 72 h by roughly 10%. However, this stabiliser is restricted to H PLC technology but does not work reliably with immunoassays. Lysis of blood stabilised homocysteine, but homocysteine concentrations were systematical ly lower requiring totally new reference ranges. In addition, acidic citrat e (0.5 mol/l) was evaluated, which seems to stabilise plasma homocysteine c oncentrations at ambient temperatures for several hours. However, small but systematic deviations at baseline are observed. This stabilisation procedu re does not interfere with immunoassays. Because immunoassays will be the f uture method of choice for robust and easy to perform homocysteine measurem ents, because they easily allow the analyses of high sample numbers, homocy steine stabilisation in whole blood is still an important matter. It must b e solved before homocysteine determinations are introduced as a general scr eening for vascular risk factors in non-specialist laboratories.