Un nuovo metodo per il dosaggio reflettometrico della emoglobina totale

Playing a crucial role in the diagnosis of many haematological disorders, total haemoglobin assay is one of the most frequently performed tests. A new dry chemistry method for the determination of haemoglobin concentration in human whole blood has been devised in our laboratory. After: a preliminary screening on a number of microporous membranes made of different materials (nylon, cellulose, polyvinylidene fluoride, nitrocellulose, cellulose acetate, polysulfone, glass fiber), a supported nylon composite-membrane,0.2/0.04 µm pore size, was chosen as the preferred matrix on the basis of its efficiency to lyse blood. The first layer (0.2 µm pore size) of the cornposite membrane exerts a strong capillary effect on plasma and the subsequent hydrodynamic flux causes the mechanical disruption of red cells and the release of their cytoplasm. The haemoglobin diffuses toward the second layer (0.04 µm pore size) and produces an unifonnely coloured disc on the surface opposite to the side where blood was applied. ln order to produce a stable haemoglobin derivative, the membrane was pretreated with a solution containing I(SCN and I(r[Fe(CN)ul, and dried. The reflectance at 556 nm of the colouted spot was evaluated at the 0.04 µm side of the membrane by the use of a laboratory reflectometer. Data obtained by analysing with this method EDTA-treated blood samples at different haemoglobin levels were compared with reference values determined on the same samples by an automatic cytoanalyzer Coulter Counter System S-Plus STIKR. Among the various equations tested to fit our results, the best linear fitting was obtained when 1/reflectance was plotted against the haemoglobin concentration. Statistical analysis gave a regression coefficient of 0.985. Assays performed using different volumes (from 10 µL to 50 µL) of blood samples showed no dependance on either the amount of applied blood or other erythrocite parameters . Time needed to reach a stable reflectance reading was as short as 50 seconds.