In vitro hematocrit measurement using spectrally encoded flow cytometry

Abstract

Measuring key physiological parameters of small blood samples extracted from patients could be useful for real-time clinical diagnosis at the point of care. An important parameter required from all blood tests is the blood hematocrit, a measure of the fractional volume occupied by the red cells within the blood. In this work, we present a method for in vitro evaluation of hematocrit based on the data acquired using spectrally encoded flow cytometry. Analysis of the reflectance confocal images of blood within a flow chamber resulted in an error as low as 1.7% in the measured hematocrit. The technique could be used as part of an in vitro diagnostic system that measures important blood parameters at the point of care.

Keywords: (170.1470) Blood or tissue constituent monitoring; (170.1530) Cell analysis; (170.1790) Confocal microscopy.

Fig. 1

Schematic illustration of the in vitro SEFC system. SLD: super-luminescence diode array. L1-L6 – lenses. LP1,2- Linear polarizers. BS – beam splitter. G – transmission grating.

Fig. 2

SEFC images at different depths without (a) and with (b) crossed polarizers that remove the glass reflection completely but also eliminate the signals from the cells.

Fig. 3

SEFC images of blood at different depths for different hematocrit levels.

Fig. 4

(a) Mean images brightness (µ) for different HCT levels and imaging depths. (b) Mean brightness for different HCT levels at z = 14 µm. (c) Measured vs. control HCT of eight blood samples using the best fit equation in (b). (d) Mean image brightness as function of depth for different HCT levels. (e) HCT vs exponential decay rate. Hollow diamonds represent the lowest HCT levels that were excluded from the linear fit. (f) Measured vs. control HCT of eight samples using the decay rate equation in (e). SEE - Standard error of estimate.

Fig. 5

Standard deviation (a) and coefficient of variation (b) of the SEFC image brightness for different HCT and imaging depths. (c) Measured vs. control HCT of eight samples using the coefficient of variation fit equation for 14 µm imaging depth. SEE- Standard error of estimate.

Fig. 6

(a) Mean image brightness vs. the coefficient of variation for different HCT levels. The data point corresponding to the shallowest depth (10 µm) is marked by hollow triangles. Depth intervals between neighboring points are 2 µm. (b) Three-dimensional surface representing HCT as function of μ and c_v for the 22 μm - 44 μm depth range. (c) Measured vs. control HCT of eight samples using the fit equation in (b) for a single image captured at an arbitrary depth. (d) Same as (c), with averaging over multiple images acquired at different depths with 2 μm intervals. SEE - Standard error of estimate.