PHOTODYNAMIC THERAPY AND GREEN LASER BLOOD THERAPY
Results: It was established that low level laser therapy when used on human blood in vitro, affects the rheology of erythrocytes and leucocytes. It was observed that it changes the erytherocytatory, leucocytatory, BSR, aggregability indices of blood.
Conclusions: Thus it was concluded that low level laser therapy can affect the physical as well as chemical properties of blood cells which is not only helpful in preservation of blood but also in revitalizing the physically and chemically stressed erytherocytatory membranes. It was determined that the laser therapy decreases the viscosity of blood thus increasing the electrophoretic mobility of erythrocytes.
CHAPTER I: INTRODUCTION
BACKGROUND OF THE STUDY
Laser irradiation, especially the low power laser has been shown to have positive effect on living tissues and as such have been used to treat various pathological conditions such as tissue repair and wound healing. Intrailuminal irradiation of atherosclerotic plaques with laser beam has been shown to recanalize obstructed arteries (Abela et al 1982).
In addition, four important effects of low level laser light have been highlighted in the scientific literatures which are tissue regeneration, reduction of inflammation, pain relief and immune system enhancement. In the light of the benefits realized through low power laser irradiation, research on the biostimulatory effects of this therapy on rheological properties of blood cells is an area of great interest for hematologists. This will ameliorate the use of this method and further generate more insight on the impact of the therapy in the rheological properties of blood cells.
The underlying mechanism of the therapy is that when blood is irradiated with low level laser in an oxygen rich environment, porphyrins absorb energy from photons and transfer this energy to the surrounding oxygen molecules. Porphyrins are a component of hemoglobin which carries oxygen to various tissues of the body. When porphyrins are not a component of hemoglobin anymore, as in preserved blood, they absorb light.
The term Photodynamic therapy denotes the in vitro therapy of blood cells which is done to change the rheological properties of blood cells, when preserved for transfusion purposes. It involves the use of photoactive drug (photosensitizer) and light which is typically visible or infrared light. When light is absorbed by porphyrin molecules, a chemical reaction is initiated which leads to direct and indirect production of cytotoxic radicals and singlet oxygen (Maiya 2000; Brancaleon and Moseley 2002) . These toxic chemicals once formed, damage the proteins, lipids, nucleic acids and many other particles of blood without causing any damage to the surrounding irradiated blood components which are **PS-free. For example viruses can be killed in whole blood without destroying blood components. (Henderson and Dougherty 1992; Sitnik, Hampton et al. 1998; Maiya 2000; Castano, Mroz et al. 2006; Morton, McKenna et al. 2008; Wilson and Patterson 2008) .
Weber in 2005 used a green laser light for the first time for intravascular blood treatment. The basic idea was to increase the energy assimilation of blood by the absorption of green laser light as a complementary color to red light (and red color of erythrocytes). With intravascular positioning of the red light catheter, it was observed that a red spot shines spontaneously through the skin, when the red light was switched on, due to the light reflecting property of hemoglobin. Whereas, no green spot appeared on the skin by switching on a green laser light with a wavelength of 536 nm, as the laser light of this wavelength is almost completely absorbed by hemoglobin. This laser irradiation therapy was introduced for the first time by Weber for the treatment of many diseases. A comparative study between red and green laser light was also conducted, by treating those patients with green laser irradiation who had already been treated with red laser previously.
After this development in the field of low level laser therapy, 20 liver patients and 20 lipometabolism patients were treated with mere green laser light successfully, demonstrating more acceptable results than red light therapy. At that time the effects of green laser on the rheological properties of blood were discovered which were more beneficial than red light (Weber, Fu ganger -May 2007).
The effects of green laser irradiation on blood cells that have been observed include the absorption of green light quanta by hemoglobin and different cytochromes, katalases and peroxidases. In addition the irradiation has been observed to stimulate the electric activity of the red cells membrane potential together with activation of the membrane potential of the mitochondria.
There are many different views about the intensity of laser light that is used to treat blood in vitro. The effluence rate of laser which is used to activate the toxic radicals in the blood should certainly be lower than the damage threshold of surrounding vital tissue components. Whereas according to Fischer and Aulmann (1998) most of the time it is desirable to use the highest possible effluence rates in order to achieve maximum effects of photodynamic therapy.
STATEMENT OF THE PROBLEM
Photodynamic therapy has long been used in treatment of various conditions due to its effect on rheological properties of blood cells. However there has been insight on the beneficial and damaging threshold levels of these radiations in human blood cells
GENERAL OBJECTIVE
To investigate the effects of different levels of laser irradiation on the rheological properties of human blood in vitro using the diode laser pointer 532.
SPECIFIC OBJECTIVES
To assess the effect of laser energy on different blood cells To assess the effect of laser on the monographic changes of blood cells. Conceptual framework
Limitation and delimitations of the study Definition of the study Structure of the study Summary of the chapter
CHAPTER II: LITERATURE REVIEW
Various studies have indicated that low energy laser light is responsible for stimulation of cell division and protein synthesis (Lubart RH. Friedmann, 1993). This phenomenon is believed to be caused by light being absorbed by endogenous porphyrins and cytochromes present in the mitochondria which in turn cause an alteration in the intracellular concentrations of ATP and ADP. These biological processes are believed to be as a result of irradiation of cells (Passarella et al 1984). Blood is a biological component able to affect the functioning of the whole organism and as such intravenous laser exposure of blood (ILEB) occupies a vital position in the existing varieties of laser biostimulation (Golub et al., 2003).
MECHANISM OF ACTION OF LASER RADIATION ON BLOOD
Laser biostimulation has gained widespread practical application, however the modes of action behind this procedure remain unclear. It is believed though that upon laser radiation, erythrocytes’ hemoglobin molecules undergo conformational changes that results in an increased efficiency of oxygen metabolism (Golub et al 2003). Observation of blood samples under an electron has revealed that laser exposed cells undergo cell membrane denaturation (Abela et al., 1985).
Upon laser exposure to a cell, electromagnetic field from the laser radiation interacts with the cells’ intrinsic electromagnetic fields altering the cell’s field charge by redistributing it. (Salyaev et al., 2003). Low energy laser radiation activates metabolic enzymes and protein biosynthesis and promotes tissue regeneration and microcirculation (Salyaev et al., 2003).
CHAPTER III: MATERIALS AND METHODS
MATERIALS
During this research diode laser pointer 532 was used as the irradiation source with a voltage of 532nm and a high current of 100mw. Unlike ordinary light, laser is a high energy device and emits photons on only one direction.
The apparatus used to measure values of the irradiated and non-irradiated blood samples was the automated hematology analyzer machine (Sysmex XE -2100). It is a machine that is used for measuring various chemicals and other properties in many biological samples. It is a quick method and requires almost no individual assistance. This method has many advantages. For example the blood samples can be read in batches or otherwise solely if needed. Thus it assists in research sample readings where a large number of samples are to be read. In blood analysis, the automate hematology analyzer machine is used to measure complete blood count, erythrocyte sedimentation rate and or coagulation profile.
For measurement, dilute samples of blood were passed through an aperture. Electric current was also passing through it. The flow of current brought a variation in the impedance between the ends. Then a lytic reagent for breaking red blood cells was added in the solution. It did not affect the white blood cells and platelets leaving them intact.
BLOOD COLLECTION
This research was conducted on one hundred blood samples which were collected under the guidelines of National Medical Research from pathology laboratory in Pulaupinang General Hospital. This study was approved by the national institute of health for conducting research in the Ministry of Health Malaysia and also by the Committee of Medical Research and Ethics. Hundred pathological samples, 5ml each, were obtained from healthy and non healthy adults (all above 18 years age) with different medical histories. The samples were divided into four groups to determine the effect of different levels of laser therapy. After collection of the blood samples, an anti-coagulant Potassium ethylenediaminetetreacetic acid (K2/EDTA) (Vacationer, BD Franklin Lakes NJ USA), was added to prevent coagulation. It is a poly amino carboxylic acid which has both in vivo and in vitro applications. It is the most widely used anticoagulant for complete blood count. Each blood sample was further divided into two halves (2.5ml each) and one of them was irradiated whereas the other was kept as control. This control was done to check for blood damage due to the irradiation system.
LASER IRRADIATION
All the four major groups were irradiated with Green diode laser with a wavelength of 532 nm at 100Mw in a continuous wave mode, with divergence < 1.5mRad, Beam Mode (TEMoo), Beam diameter at aperture
