Hematocrit determination in small bore tubes by differential spectrophotometry

Herbert H. Lipowsky, Shunichi Usami, Shu Chien, Roland N. Pittman

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A method for the measurement of the hematocrit of blood flowing in small-bore glass tubes (HT) 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 HT and luminal diameter (DT). 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 (ODabs), which is linear with HT, 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, DT. 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)ccell × HTDT, where ccell 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 HT in these tubes revealed an accuracy of 11.6% (rms error) between values of HT 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.

Original languageEnglish (US)
Pages (from-to)42-55
Number of pages14
JournalMicrovascular Research
Issue number1
StatePublished - Jul 1982

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Cardiology and Cardiovascular Medicine
  • Cell Biology


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