Hematocrit determination in small bore tubes by differential spectrophotometry

A method for the measurement of the hematocrit of blood flowing in small-bore glass tubes (H_T) is examined which relies upon a linear relationship between the difference in optical densities (ΔOD) at two isobestic wavelengths for oxy- and deoxyhemoglobin (546 and 520 nm) and the product of H_T and luminal diameter (D_T). The foundation for the technique has been shown to be firmly based upon the principles that: (1) The total optical density of blood flowing in a smallbore glass tube may be expressed as the sum of a pure absorption term (OD_abs), which is linear with H_T, and a nonlinear scattering term (B); and (2) at two closely spaced wavelengths, the scattering terms are approximately equal such that subtraction of the two total optical densities results in a term linearly proportional to the product of the difference in molecular extinction coefficients (Δε), hemoglobin concentration, and path length, D_T. These relationships were examined for the case of blood flowing in tubes ranging from 40 to 70 μm in luminal diameter and yielded a linear relationship of ΔOD = (ε₅₄₆ - ε₅₂₀)c_cell × H_TD_T, where c_cell is the mean corpuscular hemoglobin concentration. The slopes of these calibration curves, obtained for hemoglobin solutions and red cell suspensions, agreed with macrospectrophotometric values of Δε to within 2 and 12%, respectively. Simulation of a blanket application of the method to the measurement of H_T in these tubes revealed an accuracy of 11.6% (rms error) between values of H_T computed from the regression curve and those obtained by centrifugation. It is also shown that by choosing isobestic wavelengths with extinction coefficients in the ratio of 2:1, the scattering term may be computed from the relationship B = OD₅₂₀ - ΔOD; and that variations in B attendant to red cell shape changes, as for example in the case of the sickling of Hb SS cells during deoxygenation, may be easily evaluated.