BSu3A.38.pdf Biomedical Optics and 3D Imaging OSA 2012

Response to Optical Trapping by Red Blood Cells (RBCs)

from a Transfused Sickle Cell Patient

D. Erenso1, A. Pellizzaro

2, G. Welker

1, O. Mohammed

1, A. Farone

2, and M. Farone

2

Department of Physics & Astronomy1, Department of Biology2, Middle Tennessee State University, P. O. Box 71, Murfreesboro, Tennessee

37132; Tel.:615-494-8853; Fax: 615-898-5303;

E-mail:derenso@mtsu.edu

and M.d.P. Aguinaga3,4

Department of Obstetrics and Gynecology3 and, Meharry Sickle Cell Center4, Meharry Medical College Nashville,

Tennessee 37208; Tel.:615-327-6591; Fax:615-327-6593

Abstract: The response of RBCs from a transfused sickle cell anemia (Hb S and Hb A) patient

has been studied in comparison with RBCs from a healthy person (Hb AA) when directly trapped

by a laser. OCIS codes: (170.0170) Medical optics and biotechnology; (350.4855) Optical tweezers or optical manipulation

1. Introduction

Normal RBCs are responsible for delivering oxygen to body tissues by squeezing through capillaries narrower than

the normal diameter of a RBC. However, this deformability of RBCs is drastically diminished in Sickle Cell Disease

(SCD), where the RBCs change their shape under deoxygenating conditions and loose deformability occluding the

blood vessels in the microcirculation [1]. Laser tweezers [2] have been used to study these properties of RBCs in

order to measure the therapeutic efficacy of Hydroxyurea which is commonly used drug to treat SCD [3]. In this

study, we report the response of RBCs from a transfused sickle cell anemia (SCA) patient when directly trapped by

an intensity gradient optical trap in comparison to RBCs from a healthy person with normal hemoglobin.

2. Experimental Method

Blood from a transfused SCA patient was obtained from the Meharry Sickle Cell Center, Nashville, Tennessee. A

biological material transfer agreement was processed between both institutions involved, MTSU and MMC. The

hemoglobin type assessment was done by the Ultra2-High Performance Liquid Chromatography (HPLC) from

Trinity Bio-Tech (Kansas City, Kansas). The sample contained Hb S (53.68%), Hb A (39.72%), Hb A2 (3.60%) and

Hb F (3.0%). The patient had been transfused with packed RBC’s from a normal hemoglobin donor.

Our first measurement shows the RBC size distribution of both, the transfused SCA patient blood and normal

hemoglobin blood from healthy donor. The size distribution for these two samples as measured by the mean

diameter of the cells is shown by a histogram in Figure 1. As one clearly sees from Fig. 1 the size of RBCs from the

transfused SCA patient blood has a wider range (~4m-9m) as compared to those from the normal hemoglobin

blood sample (~4.8m-7.2m). The average diameter of the RBCs from the transfused SCA patient blood is 6.6m

with a higher standard deviation of 0.9m whereas the RBCs from the normal hemoglobin blood sample is 6.2m

with less standard deviation of 0.5m. The higher average size with a higher standard deviation is the result of the

simultaneous presence of the abnormal sickle hemoglobin cells (which tend to be bigger in size) and the normal

hemoglobin RBCs that came from the transfusion treatment with normal hemoglobin packed RBCs that the patient

had received.

The normal individual RBCs transfused into the SCA patient are expected to have nearly the same size as the

RBCs from the healthy individual blood sample. Therefore, these cells, most likely, are those cells that have size

range in the overlapping region of the two size distributions in Fig. 1. The target to our investigation has been on

these cells in anticipation to investigate whether transfused RBCs behave the same or differently in a foreign

environment than those RBCs in their native environment. The behavior we investigated focused on the responses of

the cells when they are trapped by an intensity gradient optical trap, kept inside the trap for fifteen seconds, released

and fully relaxed, and re-trapped again by a stronger trap and released back again. We used well collimated and

highly focused infrared (1064nm) laser to form the intensity gradient optical trap. During the process the range of

the power of the laser at the trap location, which is right outside the objective lens of the microscope (IX 51

Olympus Inverted microscope) used in our laser-tweezer set-up, was ~8-80 mW.

BSu3A.38.pdf Biomedical Optics and 3D Imaging OSA 2012

The measurement on RBCs from the healthy individual was conducted first. The blood sample (obtained by

finger pricking using a micro-lancet device) was diluted with Fetal Bovine Serum (FBS) at 1:1000 dilutions. The

diluted blood sample was placed on a well-slide on an inverted microscope which is equipped by 1.25 numerical

aperture objective lens used to focus the collimated laser beam to form the trap. The microscope is also equipped

with a digital camera interfaced to a computer which displays the live image of the cells in the well slide. We took

three consecutive images of the cell when free, lying on, or floating near to the bottom of the slide with its platelet

side (its flat and wider side). We then open the gate at the laser port of the microscope. The cell instantly flips and

gets trapped with its platelet side parallel to the direction of the laser beam propagation. We immediately raised the

trapped cell about twelve micrometer from where it was by raising the objective lens to avoid any effect caused by

contact of the cell with the bottom of the slide. Three consecutive images with five seconds intervals were taken

before the cell was released from the trap.

Figure 1. The size distribution of free RBCs as measured by the mean diameter of individual cells. RBCs from a transfused SCA patient are

shown in red and RBCs from a normal hemoglobin individual are shown in blue.

After the cell is released from the trap we took images of the cell as it continues to relax and recover its original

size and shape. These images were taken in fifteen seconds interval for period of two minutes and fifteen seconds.

During this period since the cell continues to drift towards the bottom of the slide, the objective lens had to be

adjusted to keep the cell on the focal plane. By the end of the relaxation period the cell nearly recovers its original

size and shape prior to trapping. Then after less than five minutes of transition period, we re-trap the cell with a trap

set at higher power. We began this procedure with the trapping laser power at 30mW [~8mW at the trap (cell)

location] and repeated it for several higher power until we reach the state when the cells integrity are being

compromised which was about 300mW [~80mW at the trap location]. For the sake of convenience the power is

measured at a position near to the microscope laser port which is about 38 cm behind the back of the objective lens.

The exact same procedure is performed for the RBCs from the transfused SCA patient. We have conducted these

measurements for ten different RBCs in each blood samples.

3. Data Analysis and Result

The result of our measurements focusses particularly on comparative analysis in relative size and shape changes due

to direct laser trapping of RBCs from the healthy individual and RBCs from the transfused SCA patient. We will

also include a preliminary result for the relaxation rate for RBCs from the transfused SCA patient.

The images of the cells are analyzed using image processing, enhancement, and analysis software called Image-

Pro Plus 6.2 (Media Cybernetics). Using Image-Pro Plus 6.2 we measured the mean diameters of each cells when it

is free prior to trapping (d0) and inside the trap (d) from the corresponding images. We then calculated the average

values for d and d0 at a given power setting from the measurements we made for the ten different cells. Using these

two average values we determined the relative change in the mean diameter of the cell at a given power using %

change in diameter= ((d0-d)/d0)100%, The results are shown in Fig. 2a in which blue represents the result for

RBCs from a normal hemoglobin individual and red is for transfused RBCs in a SCA patient. Each of these data

points represents the average value of ten cells measurements at the specified power. The bars represent the standard

deviation in values to those measurements. These results show that the cells have reduced size inside the trap in

either case. That is mainly because of two reasons. The first one is that the cell begins to contract towards the center

of the trap as the power of the laser trap become stronger due to increase in intensity gradients. The second is that

since the cell prefers to be in a minimum energy state, it flips and the image that the camera took is the side view

which has very narrow transverse diameter which results in significant decrease in the measured mean diameter. As

BSu3A.38.pdf Biomedical Optics and 3D Imaging OSA 2012

it contracts it also begin to flip back to its original state and also slightly expand along its narrow transverse

dimension which eventually results in a sort of spheroid shape at higher power. For low power the percentage

change for the RBCs from the transfused SCA patient blood is higher than the RBCs from the normal hemoglobin

individual blood sample. However, this behavior essentially disappears at higher powers due to the extreme

contraction of the cell from the high intensity gradient resulting from the high power.

Figure 2. (a) The percentage change in the mean diameter of the cell inside the trap relative to the mean diameter of the cell when free RBCs

from a healthy donor are shown in blue, and RBCs from the transfused SCA patient blood in red. (b) The percentage change in the mean diameter

of the cell as it relaxed relative to free cell (solid) and relative to trapped cell (dotted). Red is at 30mW and black is at 230mW.

The relaxation rate for the RBCs from the transfused SCA patient for two different powers of the trap is shown

in Fig. 2b. These relaxation rates are analyzed in terms of relative changes in the mean diameter as function of time.

Following a similar procedure we calculated the relative change in the average diameter, % change in diameter=

((d0-d)/d0)100%. However, for this relative changes d is the measured mean diameter of the relaxing cells in 15

seconds intervals measured immediately after the laser power is cut off. For d0, we used the average mean diameter

for the free cell, like previously discussed, but we also used the mean diameter of the cells when it was trapped. In

Figure 2b Red demonstrates the relaxation rate for the cells at low power (30mW) while black is at high power

(230mW). Each data point represent the % difference calculated using the average of the mean diameters (for d and

d0) for ten different cells. The data points best fitted by the solid curves represent when d0 is the diameter of the free

cells prior to trapping and those best fitted by the dotted curves represent when d0 is the diameter of the trapped

cells. For the solid curves zero value show the cells has fully recovered its size and shape and high negative values

show the cells have maximally contracted relative to its free prior to trapping sizes. For the dotted curves, however,

zero value shows maximal contraction and high negative values show maximal relaxation or full recovery relative to

its corresponding size inside the trap. The relaxation rates of the cells as measured relative to its corresponding sizes

inside the trap (the dotted curves) at 30mW and 230mW, interestingly, demonstrate that the cells that have been

released from a high power trap on the average recover from its deformation relatively faster than those cells less

deformed at the lower power trap.

4. Conclusions

We have studied how transfused RBCs with normal hemoglobin in SCA patient respond to direct laser trapping in

comparison with RBCs from a normal individual. The results show that the relative size change is higher for the

RBCs transfused in a SCA patient than those in a normal individual. The relaxation rate conducted for the transfused

RBCs, interestingly; shows that the cells released from a high power trap appear to relax relatively faster than those

released from a low power trap. This suggests that the change in the laser power changes the temperature of the FBS

(and the cells) which could trigger different behavior in the cells that affected the relaxation rates.

5. References

[1] C. Madigan and P. Malik “Pathophysiology and therapy for haemoglobinopathies; Part I: sickle cell disease,” Expert Rev. Mol. Med. (8) 1-23 (2006).

[2] A. Ashkin, “Applications of laser radiation pressure,” Science 210 (4474), 1081-1088 (1971).

[3] M. M. Brandao, A. Fontes, M. L. Barjas-Castro, L. C. Barbosa, F. F. Costa, C. L. Cesar, and S. T. Saad, “Optical tweezers for measuring red blood cell elasticity: application to the study of drug response in sickle cell disease,” European J. of Haematology. 70 (4), 207-211 (2003).

(a) (b)