Deformability analysis of sickle blood using ektacytometry

Abstract

Sickle cell disease (SCD) is characterized by decreased erythrocyte deformability, microvessel occlusion and severe painful infarctions of different organs. Ektacytometry of SCD red blood cells (RBC) is made difficult by the presence of rigid, poorly-deformable irreversibly sickled cells (ISC) that do not align with the fluid shear field and distort the elliptical diffraction pattern seen with normal RBC. In operation, the computer software fits an outline to the diffraction pattern, then reports an elongation index (EI) at each shear stress based on the length and width of the fitted ellipse: EI=(length-width)/(length+width). Using a commercial ektacytometer (LORCA, Mechatronics Instruments, The Netherlands) we have approached the problem of ellipse fitting in two ways: (1) altering the height of the diffraction image on a computer monitor using an aperture within the camera lens; (2) altering the light intensity level (gray level) used by the software to fit the image to an elliptical shape. Neither of these methods affected deformability results (elongation index-shear stress relations) for normal RBC but did markedly affect results for SCD erythrocytes: (1) decreasing image height by 15% and 30% increased EI at moderate to high stresses; (2) progressively increasing the light level increased EI over a wide range of stresses. Fitting data obtained at different image heights using the Lineweaver-Burke routine yielded percentage ISC results in good agreement with microscopic cell counting. We suggest that these two relatively simple approaches allow minimizing artifacts due to the presence of rigid discs or ISC and also suggest the need for additional studies to evaluate the physiological relevance of deformability data obtained via these methods.

Keywords: Laser diffraction ellipsometry; diffraction pattern; irreversible sickled cells; sickle cell disease; sickle erythrocytes.

Fig. 1

Schematic intensity pattern of diffracted laser light for sickle cell disease red blood cells at high shear (e.g., 50 Pa). The effects of both techniques used herein are illustrated: altering the image height via changing camera aperture or changing gray level (i.e., light intensity) used for fitting. The distorted diffraction pattern and the resulting EI are shown for control height or a low gray level (left drawing) with the pattern and resulting EI shown for reduced image height or higher gray level (right drawing).

Fig. 2

Representative elongation index (EI) versus shear stress (SS) results for normal RBC from a single donor obtained using different aperture settings and hence different image heights (control, 15% and 30% reduction). No differences were detected between these three heights.

Fig. 3

Representative elongation index (EI) versus shear stress (SS) results for normal RBC from a single donor obtained using different gray level settings and hence different light intensities for fitting; an increased gray level indicates a higher light intensity. No meaningful differences were observed in the deformability results after changing the fit gray level.

Fig. 4

Diffraction images and resulting fitting of the images (bright lines) for the same SCD red cell population at two levels of shear stress and two image heights: (A) and (B) are both at 0.5 Pa showing control image height (A) and 30% reduction (B); (C) and (D) are both at 50 Pa with (C) at control height and (D) at 30% reduction. Note the horizontal bulge and poor fitting at control height (C) and the minimal bulge and improved fitting at 30% reduction (D). (Colors are visible in the online version of the article; http://dx.doi.org/10.3233/BIR-140660.)

Fig. 5

Representative elongation index (EI) versus shear stress (SS) results for sickle cell disease RBC from a single donor obtained using different aperture settings and hence different image heights (control, 15% and 30% reduction). Note that at stresses ≳2 Pa there is an inverse relation between EI and image height.

Fig. 6

Representative elongation index (EI) versus shear stress (SS) results for sickle cell disease RBC from a single donor obtained using different gray level settings and hence different light intensities for fitting; an increased gray level indicates a higher light intensity. Note that at ≳2 Pa an increased gray level yields a higher EI.

Fig. 7

The effects of percentage ISC in sickle cell disease RBC populations on the numerical difference of EI_max between control and 15% reduced heights. Percent ISC was determined by optical microscopy for 1,000 cells per subject. Data are for 23 SCD subjects; the straight line was determined by linear regression (R² = 0.8683, p < 0.001).