Oxyhemoglobin measurement of whole blood specimens in a silicon microfabricated cuvette

The purpose of this study was to develop a miniaturized CO- oximeter for hemoglobin derivative measurement using microfabrication technology. A microcuvette (volume equals 507 nl) was fabricated for analysis of percent oxyhemoglobin (O2Hb%) in whole blood. A cuvette of 50 micrometer pathlength produced optimal absorbance sensitivity to changes in O2Hb%. The pressure differential for a nominal blood flow rate of approximately 1 microliter/second was 4.1 kPa (16.6 in water, 0.6 psi). Entrained bubbles were easily discharged at these pressures. Spectral measurements were made using an ocean optics miniaturized spectrophotometer (500 - 700 nm). A fiber optic probe with one receiving and six emitting fibers (200 micrometer core and 0.22 NA) was used for spectral measurement. Heparinized fresh blood from a healthy volunteer was tonometered with N2, CO2, and O2 mixtures to produce six samples with O2Hb% from 22 - 97%. Chemometrics was used for data analysis. The second derivatives of spectra were taken to eliminate baseline changes caused by RBC light scattering. Indirect calibration by principal component regression was applied to the second derivative. Four factor cross validation showed a correlation coefficient of 0.9994 between measured O2Hb% of lysed blood using an OSM3 CO- oximeter (Radiometer America, Ohio) and whole blood using the microfabricated cuvette. The linear relationship is: O2Hb%micro-cuvette equals 0.8411% plus 0.9882 multiplied by O2Hb%OSM3. We conclude that O2Hb% measurement on unlysed whole blood using a silicon microfabricated cuvette is practical and that results are similar to traditional CO- oximetry.