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41BIOPHOTONICS OF BODY FLUIDS FOR CLINICAL APPLICATIONS: MICRO-RAMAN STUDY OF
BLOOD CELLS IN INTRAVENOUS FLUIDS
SANTHOSH CHIDANGIL, MITHUN N., REENA V. JOHN, SPHURTI S. ADIGAL, NIDHEESH V.R. AND
JIJO LUKOSE
Centre of Excellence for Biophotonics Department of Atomic and Molecular Physics, Manipal Academy of Higher Education Manipal, Karnataka, India-576104 Email:santhosh.cls@manipal.edu
Investigation of body fluids is of paramount importance since it carries a pool of biological markers, which can reflect the health status of humans. Technological advancements in the field of biophotonics offer highly sensitive, objective and rapid detection of abnormal health conditions from body fluid analysis. Raman spectroscopy has been widely regarded as reliable spectroscopic tool for the characterization of body fluids due to its minimal interference imposed by aqueous environment. Raman Investigation of blood cells under physiological condition is restricted due to Brownian motion of micron sized cells. In view of this, spectroscopic characterizations of live cells in intravenous fluids has been performed using a custom built Raman Tweezers, which involves the optical arresting of cells with the aid of tightly focused laser beam.
Introduction
Raman spectroscopy technique is considered as a widely accepted methodstechnique for studying biological samples [1]. The technique has been in use for various fields like molecular structure analysis, biochemical research, forensic science, disease diagnosis, chemical analysis etc. [2, 3]. Raman spectroscopy uses inelastic light scattering to provide enough details about the molecular makeup of materials, such as the specific functional groups found in biological and chemical analytes [4]. In Raman spectroscopy techniques the monochromatic light radiation is allowed to interact with the sample and the scattered light will detected by the spectrograph/spectrometer. The most of the light will be elastic scattered (Rayleigh scattering) and the small portion inelastic scattered (Raman scattering) [5]. The Raman signals (Stokes Raman scattered radiation) collected using an edge filter after removing the Rayleigh scattered radiation can be used as a "fingerprint" for the identification of the target molecular species.
Raman spectroscopic investigations of single live cells were possible with the aid of optical tweezers [6]. The Raman spectroscopic technique was combined with the Optical tweezers to form Laser tweezers Raman spectroscopy. The spectroscopic investigations of live cells were very difficult due to the Brownian motion of the cells from the liquid media. The hurdle was overcome by the invention of Optical tweezers technique by Arthur Ashkin [7, 8]. An optical tweezers technique consist of a tightly focused laser beam to arrest the moving cells for Raman measurements. Different research groups investigated the biochemical changes in Red Blood Cells (RBCs) as a function of temperature, the interaction of cells with various external agents (chemicals), laser irradiation, and so on [9-12].
Intravenous fluids (IV fluids) are liquids delivered straight into the veins of patients who are unable to meet their daily demands through food or drink. Water, salts, and sugar are all present in the intravenous fluids. There are different intravenous fluids in daily use under healthcare settings. Several groups are conducting research on intravenous fluid infusion and the after effects [13, 14]. Coburn H. Allen et al. conducted a study to compare the effects of normal saline and Plasmalyte-An intravenous fluid is a replacement in children suffering from moderate to severe dehydration due to acute gastroenteritis (AGE). From this study they have concluded that the plasmalyte-A infusion is more effective than normal saline infusion [15]. Scott A. Kirkley et al. conducted a comparative research based on Red Blood Cell washing with plasmalyte-A and normal saline. They found that RBCs held for 10 to 39 days and washed with plasmalyte-A had less hemolysis and a longer storage time than those stored in normal saline [16]. Neil Blumberg et.al conducted an experiment based on the impact of Normal saline and plasmalyte-A on sickle cell and normal RBCs [17]. The RBCs incubated (in-vitro) with normal saline was showing more hemolysis than Plasmalyte-A. They have also mentioned that the normal saline increases the rate of renal failure in critically ill patients. So the studies related to the impact of intravenous fluids on the single live RBCs have paramount importance. The microscopic images and quantitative phase images of the RBCs suspended in blood plasma and intravenous crystalloid fluids were also discussed in this current work.
Materials and methods
The samples were collected from the Blood Bank, Kasturba Medical College, Manipal with the permission of Institutional ethics committee. The fresh and healthy blood samples collected from the Blood bank was centrifuged for 5 minutes with 3000 rpm for obtaining packed RBCs. For the current study the packed RBCs were suspended in different intravenous crystalloid fluids (Normal saline 0.9%, Plasmalyte-A, Ringer lactate, Hypertonic saline 3%, Hypotonic saline 0.45%, Dextrose normal saline (DNS) and Dextrose 5%). The current experiment was carried out for studying the effect
of intravenous crystalloid fluids on single live RBCs. Because blood plasma maintains physiological conditions to a large extent, RBCs suspended in blood plasma were used as a control. The large dilution of packed cells in the intravenous crystalloid fluids prevented multiple cell trapping (0.5 ^l packed RBCs in 1 ml of crystalloid fluids). The experiments were conducted using home-built Raman tweezers spectroscopy setup shown in Figure 1. The laser beam with the wavelength of 785 nm (Star bright diode laser, Torsana laser tech, Denmark) was used for both trapping as well as the excitation of the live RBCs suspended in intravenous crystalloid fluids. A 100X oil immersion microscope objective was employed to create a tightly focused laser beam for trapping a single living cell (Nikon, plan fluor, Japan). The back scattered signals from the sample were collected using the same 100X microscope objective, but the Rayleigh scattered signals were blocked using an Edge filter. The Raman signals were directed to the spectrometer (iHR320, Horiba Jobin Yvon) with liquid nitrogen cooled CCD (Symphony 1024 x 256-OPEN-1LS). All the spectra were recorded from different RBCs with 3 mW laser power for avoiding the photo-damage of the cells. The exposure time and accumulation number for each spectra were 60 seconds and 2 respectively.
Figure 1. The schematic diagram of the home-built Laser tweezers Raman spectroscopy setup. Results and discussion
The Raman spectra of single live RBCs suspended in different intravenous crystalloid fluids and blood plasma were shown in Figure 2. The RBCs suspended in blood plasma is considered as the control for the study. The arrows given in the particular peak positions indicating the intensity changes of the Raman bands. RBCs suspended in hypertonic saline, hypotonic saline, Dextrose 5%, and DNS showed notable Raman band intensity changes. The relative intensities of oxygenation marker peaks have visible changes. The Raman bands at 565 cm-1 (Fe-O2 stretch region), 1222 cm-1 (Methine CH deformation region), 1561 cm-1 (Spin marker region) and 1636 cm-1 (spin marker region) have decreased intensity in all crystalloid fluids. The deoxygenation marker bands corresponding to 1209 cm-1 (Methine CH deformation region) and 1544 cm-1 (spin marker region) have higher intensity in all intravenous crystalloid fluids. Similarly the intensity of 1397 cm-1 (pyrrole deformation band) were decreased in all the crystalloid fluids mainly in hypotonic saline 0.45% and dextrose 5%. RBCs suspended in a hypotonic saline solution had a low overall spectral intensity. The decreased intensity of porphyrin breathing mode (752 cm-1) indicate the hemoglobin depletion of RBCs in hypotonic saline 0.45%. The oxygenation marker band intensities of RBCs treated with Plasmalyte-A, Normal saline (0.9%) and Ringer lactate were also decreased.
Figure 2. The Raman spectra of single live RBCs treated with different intravenous crystalloid fluids.
The microscopic images of RBCs treated with different intravenous crystalloid fluids were given in Figure 3. The images were captured using 100X oil immersion microscope objective. In blood plasma, the morphological changes of RBCs were not observed, which indicate that the blood plasma is acting as a buffer for the cells. The optically trapped RBC was shown in the red circle. Due to the higher flexibility of the RBC after trapping the cell was flipped and looks like a dumbbell shape, this shape change was observed in RBCs suspended in blood plasma, plasmalyte A, Ringer lactate and normal saline. The RBCs suspended in Plasmalyte-A was showing exactly similar morphological structure that of blood plasma. The RBCs were intact in plasmalyte-A. In the case of Ringer lactate and normal saline the morphology of some RBCs were started to change. The cells in normal saline was slowly changing into echinocyte stage, but the trapped RBC was showing exactly similar features as that of blood plasma. Thorn like projections were started to appear on the surface of some RBCs. The morphology of cells suspended in hypertonic saline was completely changed into echinocyte. The thorn like projections were easily visible on the cell surface. The RBCs became spherocytes in dextrose 5% and hypotonic saline 0.45%. The cells were bulged and the discoid shape of the cells were disturbed. In the case of spherocytes the optically trapped cells were not flipped, which shows that the deformation ability of the cells were reduced. These morphological changes will adversely effect the cytoskeleton structure of the RBCs and this may effect the deformability of the cells.
Blood plasma Plasmalyte A Ringer lactate Normal saline (0.9%)
DNS Hypotonic saline 0.45% Hypertonic saline 3% Dextrose 5%
Figure 3. The microscopic images of RBCs treated with blood plasma and different intravenous crystalloid fluids.
The quantitative phase images of the RBCs suspended in different intravenous crystalloid fluids were captured using d'Bioimager (d' Optron) for studying the morphological variations of the cells. A microscope objective of 50X was used to magnify the pictures exhibited in Figure 4. Usually, the normal RBCs were discoid shape, the cells suspended in blood plasma also have a discoid shape and there is no morphological deformation of the cell. A shows almost similar morphological behavior to that of the cells in blood plasma, the cell membrane damage and other deformations were not seen in the image. The discoid shape of the cell was maintained in Plasmalyte-A. The RBCs suspended in normal saline show some projections or specular structure, indicating that the RBC is morphologically shifting to the echinocyte stage, which will result in a reduction in the cell's surface area and volume. The deformation ability of the cell also reduced in echinocyte. The specular structures formed on the membrane surface in presence of hypertonic saline are evident in Figure 4. Comparing to normal saline the echinocyte formation of RBC in hypertonic solution was faster, after suspending the cells in the solution within one or two seconds the discocyte will became echinocyte. The RBCs in Dextrose 5%, hypotonic saline and ringer lactate shows spherocyte formation. The center portion of the RBCs were bulged due to the effect of these intravenous fluids. The RBCs in Dextrose normal saline (DNS) was almost looks like
stomatocytes.
Figure 4. The Quantitative phase images of RBCs treated with blood plasma and different intravenous crystalloid fluids. Conclusions
Micro-Raman combined with Optical Tweezers setup is a versatile tool to study the blood components e.g. RBCs, Platelets, WBCs etc and their interaction with external stressors. The effect of intravenous crystalloid fluids on single live RBCs were investigated using Raman tweezers spectroscopy technique. The deoxygenation tendency of the cells suspended in intravenous fluids were observed from the Raman bands. The hemoglobin depletion or denaturation was also evident from the Raman spectra of RBCs in certain intravenous crystalloid fluids. The morphological variations of the RBCs also evident from the microscopic images and quantitative phase images.
Acknowledgement:
Authors are thankful to DBT, Govt. of India for the Raman Tweezers setup and Mithun, Reena, Sphurt and Nidheesh are
for their Dr. TMA Pai Ph.D. fellowships.
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