Blood, Vol. 92 No. 8 (October 15), 1998:
pp. 2975-2977
CORRESPONDENCE
Mechanical Properties of Stored Red Blood Cells Using Optical
Tweezers
 |
LETTER |
To the Editor:
We have developed a method for measuring, simultaneously and in the
same red blood cell, membrane elasticity and viscosity using optical
tweezers.1-3 We demonstrated the capability and sensitivity
of this method by applying it to study both fresh and 35-day stored
erythrocytes preserved in CPDA1 (citrate-phosphate-dextrose-adenine). In our method, the elasticity µ was obtained by measuring the deformation of the cells when dragged at a constant velocity through the plasma fluid by optical tweezers.4 By suddenly stopping the movement mentioned above, we were able to measure the membrane viscosity 
using the time for the cell to return to its initial morphology. It has been shown that, for this return movement, the
length decreases exponentially with time, according to the expression
Lo = Lo + 
Le
t/
, where 
= /µ.5 Therefore, by measuring and µ, can be extracted. This shows how important it is to measure the return time
and the elasticity µ of the same red blood cell to obtain the
viscosity.
The optical trap consisted of a Nd:YAG laser focused through the 100×
oil immersion objective of an Olympus microscope (Olympus Optical Co,
Ltd, Tokyo, Japan) equipped with a JVC Minicam (JVC Company of America,
Elmwood, NJ) used to record the images in real time in VHS
and, finally, captured in a computer to be quantitatively and qualitatively analyzed. The model used for calculating the elasticity value assumed a parallelepiped shaped (length
Lo, width W, and negligible thickness) cell located at a
distance Z1 from the bottom of a Neubauer slip and
Z2 from the cover slip. Furthermore, it assumed a drag
force expressed by Fdrag = (WLo/Zeq)V and an elastic response given
by Felastic = µ(W/Lo)L, where Fs are the drag and elastic forces, µ is the elasticity, L is the cell length deformation, is the plasma viscosity, and
(1/Zeq) = (1/Z1) + (1/Z2) and V are the drag velocity. At equilibrium, these
two forces must cancel each other and the elongation L can be
expressed by L = [Lo2/µZeq)]V,
independent of the cell width W. The measurement of this deformation as
a function of the drag velocity can be used to extract a value for µ,
providing that the plasma viscosity , length Lo, and
Zeq are known.
The samples used were obtained from blood donors at the Hematology and
Hemotherapy Center of Campinas and analyzed in a 100-µm depth
Neubauer slip. The erythrocyte concentrate was diluted (1:500 µL) in AB plasma. The plasma viscosity = 2.19 centipoise was measured at 23°C. At least 5 erythrocytes of each bag
were submitted to 5 velocities, varying from 50 to 250 µm/s. The drag
cell image was analyzed with the Image-Pro Plus software
(Media Cybernetics, Silver Spring, MD) to obtain the deformation as a
function of velocity. At a velocity of 250 µm/s, the movement was
suddenly stopped and the images were captured with a 30-frame/second
rate to measure the length of the red blood cell as a function of time (in 1/30-second steps). Figure 1 shows
the deformed red blood cells at 0 and 35 days of storage, with
velocities varying from 120 to 210 µm/s.
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| Fig 1.
Deformed red blood cells at 35-day and 0-day storage with
velocities varying from 120 to 210 µm/s.
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The analysis of red blood cell deformation as a function of velocity in
the model showed that the points compose a straight line
(R2 = .9869). When we studied the cell length
versus time at the moment when the cell returns to the initial
morphology, we found an exponential decay curve. The Lo
value used to calculate the elasticity was the y-intercept value
obtained from the straight line; Zeq was measured by the
microscope micrometer, positioning the cell in the center of the
Neubauer slip. Table 1 shows the results obtained for both fresh and stored red blood cells.
Figure 2 is a graphic from which we can
verify the sensitivity of the method. The initial red blood cell length
Lo is plotted against the measured elasticity for nonstored
and stored cells. Considering different donors, the correlation
decreases to .83. For the 6 different fresh red blood cells (4 of the
same donor and the other from 2 different donors), the slope ranged
from 0.012 to 0.021 µm/(µm/s) and the y-intercept (cell length with null velocity) varied from 6.5 to 8.3 µm, whereas for 35 stored cells
(2 cells for each donor), the slope changed from 0.005 to 0.009 µm/(µm/s) and the y-intercept changed from 5.1 to 6.1 µm. From
these data we verified that the slope variation among different red
blood cells was around 25% for cells with same storage time, but there
was a great difference of 240% between the fresh and 35-day stored
cells. The same was observed for the size (y-intercept). There is a
clear distinction of the curves for the fresh and the stored cells.
Although the elasticity of 35-day stored cells is in the same range of
the fresh ones, there is a statistically significant difference in
their averages. Results in Table 1 show a 40% elasticity variation
from cell to cell and a 44% variation between the average for fresh
and stored cells, which is much larger than the 15% average variation
expected for a random sample. Viscosity, in contrast to elasticity, did
not present any correlation to storage time and remained practically
constant.
Loss of posttransfusion viability is the major limitation of long-term
red blood cell storage. To determine the cellular properties responsible for this loss, a number of investigators have examined changes in the various properties during storage and have reported that
a progressive loss of lipid, spherocyte form occurs and that cells
undergo a loss of deformability.6-8 When we analyzed the deformability as a function of velocity of fresh red blood cells, we
found a deformability loss of a small population of red blood cells
masked by the presence of a large percentage of normally deforming
cells. In the 35-day stored red blood cells, we found that almost all
cells had suffered an important deformability loss. Previously,
Bronkhorst et al3 demonstrated that the use of
optical tweezers is sensitive for studying shape recovery of red blood
cells.
The evidence in this study of the applicability of this technique to
monitor mechanical properties of membrane measuring elasticity and
viscosity simultaneously should help clarify the relationship between
deformability and viability of stored red blood cells for transfusion
purposes. We concluded that it is a new method that could be used as an
independent tool for the investigation of the biophysical behavior of
abnormal red blood cell membranes.
R.R. Huruta
M.L. Barjas-Castro
S.T.O. Saad
F.F. Costa
Hematology-Hemotherapy
Center
A. Fontes
L.C. Barbosa
C.L. Cesar
Instituto de
Fisica Gleb Wataghin
State University of Campinas
São Paulo,
Brazil
| |
REFERENCES |
1.
Ashkin A,
Dziedzic JM,
Bjorkholm JE,
Chu S:
Observation of a single-beam gradient force optical trap for dielectric particles.
Optic Lett
11:288,
1986
2.
Ashkin A,
Dziedzic JM:
Optical trapping and manipulation of viruses and bacteria.
Science
235:1517,
1987[Abstract/Free Full Text]
3.
Bronkhorst PJ,
Streekstra GJ,
Grimbergen J,
Nijhof EJ,
Sixma JJ,
Brakenhoff GJ:
A new method to study shape recovery of red blood cells using multiple optical trapping.
Biophys J
69:1666,
1995[Medline]
[Order article via Infotrieve]
4.
Huruta RR,
Barjas-Castro ML,
Saad STO,
Costa FF,
Cezar CL:
A new method to study mechanical properties of red blood cells using optical tweezers.
Blood
90:6a,
1997(abstr, suppl 1)
5.
Sutera SP,
Gardner RA,
Boylan CW,
Carroll GL,
Chang KC,
Marvel JS,
Kilo C,
Gonen B,
Williamson JR:
Age related changes in deformability of human erythrocytes.
Blood
65:275,
1985[Abstract/Free Full Text]
6.
Beutler E,
West C:
The storage of hard-packed red blood cells in citrate-phosphate-dextrose (CPD) and CPD-adenine (CPDA-1).
Blood
54:280,
1970[Abstract/Free Full Text]
7.
Card RT,
Mohandas N,
Perkins HA,
Shohet SB:
Deformability of stored red cells. Relationship to degree of packing.
Transfusion
22:96,
1982[Medline]
[Order article via Infotrieve]
8.
Wolf LC:
The membrane and lesions of storage in preserved red cells.
Transfusion
25:185,
1985[Medline]
[Order article via Infotrieve]
