OPTICAL TRANSMISSION
PROPERTIES OF RED BLOOD
CELLS AT HIGH OSMOLARITY
by
M. K. Swami, H. S. Patel, A. Uppal and P. K. Gupta
Laser biomedical applications and instrumentations division,
Raja Ramanna Centre for advanced technology,
Indore (MP), India-452013
INTRODUCTION
 Red blood cells being the most abundant cells in blood
dominate the light scattering behavior of blood.
 Mature red blood cells, in normal conditions are flexible
biconcave disks devoid of cell nucleus and most
organelles.
 The transmission characteristics of a suspension of Red
Blood Cells (RBCs) are determined by the combined effect
of the absorption by intracellular hemoglobin and the
scattering from the cells.
 Both these parameters are affected by the shape and
volume of the cells, in particular the measured absorbance
in the ~414 nm band for suspension of RBCs is smaller
compared with that for a hemoglobin solution of similar
concentration.
ABSORPTION SPECTRA OF RBCs
Pigment Packaging Effect
Absorber molecules when confined to
small volume may shadow each other.
The confinement also results in cross
sectional area unoccupied by the absorber.
This two effects lead to flattening of the
absorption spectra.
Jarod C. F. et. al. Opt. Lett. 29, 965 (2004).
OD
0.3
350
Suspension
0.3
50 % Packing
300
250
0.2
200
90 % Packing
0.1
0
400
0.4
Calculated Optical
density and
FWHM as a
function of
packing fraction.
150
420
440
460
Wavelength
480
500
0.0
0.2
0.4
0.6
0.8
1.0
Cross-sectional area (Absorber)/Cross-sectional area (Beam)
OD
0.4
400
Calculated
Optical
density for
the three
case shown.
FWHM
0.5
0.2
0.1
0.0
LIGHT SCATTERING FROM RBCs
 The transmission spectra of RBCs, apart form the
absorption, also depends upon scattering which results in
a background that reduces with increasing wavelength.
 The light scattering mostly arise from the hemoglobin in the
cells which is a major constituent of RBCs.
 The light scattering behavior of RBCs has been modeled
most commonly using either Mie theory or Reyleigh-Gans
approximation.
M. Hammer et. al. Appl. Opt. 37,7410(1998).
 Light scattering from RBCs approximates scattering from a
volume equivalent spherical particle.
J.M. Steinke et. al. Appl. Opt. 27,4027(1988).
EFFECT OF OSMOLARITY

Osmotic pressure results in shape deformations in RBCs.
Increasing hyper-tonicity
http://www.optics.rochester.edu/workgroups/cml/opt307/spr09/amir/Slideshow/Slideshow.html

The transmission spectrum of suspension of RBC is therefore
expected to show a significant dependence on the osmolarity of the
buffer medium.

The osmotic pressure leads to reduction in volume with a little change
in the surface area.

This is expected to result in increased pigment packaging (reduced
contribution of absorption) and increased scattering.

The change in the shape and volume of RBCs should also depend the
rigidity of cell membrane, which gets altered in various diseases
conditions.
RBCs IN HYPERTONIC MEDIUM
@ 310 mOs and @ 1700 mOs
the transmission spectra
shows the expected behavior
with prominent absorption ~
414 nm and between 540-600
nm.
Scattering contribution
decreases with increasing
wavelength.
@ 800 mOs the Scattering
contribution shows
uncharacteristic reversal and
increases with increasing
wavelength.
Transmission spectra of single scattering RBC
suspension in phosphate buffer saline (PBS) at
normal osmolarity (310 mOs) and hypertonic
osmolarities (800 and 1700 mOs).
RBCs IN HYPERTONIC MEDIUM
Slope of transmission curve (in
the range 600-850 nm ) shows
reversal of pattern at ~800 mOs
CONTINUE
The possible shape that RBCs deforms into can be used to infer the behavior.
Increasing hyper-tonicity
800 mOs
1700 mOs
Maximum force is seen by the flat surface resulting in
the displacement of hemoglobin to peripheral region
creating a circular aperture which allows smaller
wavelengths to pass more as compare to larger
wavelengths.
@ osmolarities > 1000 mOs the spherical shape
Echinocytes are formed resulting in transmission
spectra decreasing scattering with incerasing
wavelength.
EFFECT OF SERUM
Transmission spectra of single scattering RBC suspension in phosphate buffer
saline (PBS) at 800 mOs osmolarities and varying concentration of Bovine serum
albumin (BSA).
CONTINUE
Serum proteins are known to form a thin layer on the RBC membrane
and decrease the deformability with saturation effects ~ 1g/dl.
Y. Kikuchi and T. Koyama, Am. J Physiol., 247, H739 (1984).
The saturation
effect occurs at
much lower value
of concentration of
BSA as compare to
that reported for
deformability
experiments
employing other
methods.
Optical method is
more sensitive to
shape changes !!!!
Slope of transmission curve (in
the range 600-900 nm ) as a
function of BSA
RBCs FROM DIABETIC PATIENTS
Increased rigidity of RBC
membrane results in
reduced deformability of
RBCs
Transmission spectra of single scattering RBC
(from patient with fasting blood glucose level
~350) suspension in phosphate buffer saline
(PBS) for different osmolarities.
CONCLUSION
The transmission spectra of RBCs suspended in PBS
shows
anomalous
transmission
behavior
@
osmolarities ~ 800 mOs.
The anomalous light scattering behavior is a result of
circular aperture type shape acquired by RBCs .
Experiments in presence of BSA shows that increased
rigidity of cells reduces the effect of osmolarity.
Experiments on RBCs from diabetic patients
consistent with the increased rigidity of RBCs.
is
REFERENCES
1.
Jarod C. Finlay et. al., Opt. Lett. 29, 965-967 (2004).
2.
M. Hammer et. al. Appl. Opt. 37,7410(1998).
3.
J.M. Steinke et. al. Appl. Opt. 27,4027(1988).
4.
K. Tsukada et. al. Microvascular Res., 61, 239, (2001).
5.
Y. Kikuchi et. al. Am. J Physiol., 247, H739 (1984).
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