Caicai Wu

Results Obtained using A Prototype Microfluidics-Based Hematology Analyzer

Microfluidic laminate-based structures incorporating hydrodynamic focusing and flow channels with dimensions much less than 1 mm were fabricated and used to transport and analyze blood samples. Optically transparent windows integral to the flow channels were used to intercept the sample streams with a tightly focused diode laser probe beam. The size and structure of the blood cells passing through the laser beam determined the intensity and directional distribution of the scattered light generated. Forward and small angle light scattering channels were used to count and differentiate platelets, red blood cells, and various populations of white blood cells. All the blood samples used were characterized using a commercial hematology analyzer for comparison and validation purposes.

Feasibility study of the spectroscopic measurement of oxyhemoglobin using whole blood without pre-treatment

A feasibility study was carried out to evaluate a chemometrics-enhanced measurement of oxyhemoglobin concentration in whole blood without pre-treatment by lysing cellular components in the sample. Conventional in vitro multi-wavelength CO oximeters pre-process blood by sonication or detergent dilution to lyse blood cells to reduce light scattering. Two limitations result: (1) residual cell membrane fragments can seed surface biofouling and (2) dilution errors can occur. A full wavelength method using multivariate analysis in chemometrics was applied to correct the light scattering effect in the measurement of oxyhemoglobin concentration. Whole blood specimens were adjusted to different oxyhemoglobin concentrations with gas mixtures (N2, CO2 and O2). An Ocean Optics miniaturized spectrophotometer with a 100 microns pathlength optical cell was used for transmission measurements from 500 to 700 nm. Original spectra were smoothed and a second derivative transformation was performed to eliminate the baseline shift and slope changes from light scattering. Indirect calibration was applied to the second derivative spectra. Two-factor cross-validation by principle components regression on two sets of data showed r2 = 0.985 and 0.946 between predicted oxyhemoglobin concentration and those measured by an AVL 912 CO oximeter with RSD = 3.85 and 6.83%, respectively. Error analysis gave s = 2.36 x 10(-5) (RSD = 0.23%) on derivative absorbance for the spectrophotometer measurement alone. Specimen settling and specimen sampling gave imprecision on derivative absorbance of s = 6.17 x 10(-4) (RSD = 4.4%) and s = 4.52 x 10(-4) (RSD = 1.4%), respectively.

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.