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
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.
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
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.
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.
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 ~1.5, Crystal type Nd:VYO4:KTP, Power Source 1 x 3V CR2 Alkaline batteries. The power density was 509.55mW /cm2 at a distance of 6.5 cm from the laser device from blood inside the tube, and diameter of the laser spot was set 0.5 cm. Samples were irradiated in different time periods at energy effluence of 0.5j/ cm2, 1.5j/ cm2, 3j/ cm2 and 5j/cm2 for the first, second, third and fourth groups at 1, 3, 6, and 10 sec. respectively.
A diode laser pointer which was used during research was a laser pointer with a voltage of 532nm and a high current of 100mw was used. Unlike ordinary light, laser is a high energy device and emits photons on only one direction. All the irradiated and non-irradiated samples of blood were allowed to stand for about 30 minutes at room temperature, before counting was done. Blood counts were then performed both before and after the irradiation.
The method used to measure values of the irradiated and non-irradiated blood samples was automated hematology analyzer machine. 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 automated hematology analyzer machine is used to measure complete blood count, erythrocyte sedimentation rate and or coagulation profile.
COMPLETE BLOOD CELLS MEASUREMENT
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. Then these solutions were passed through another detector this getting the measurements of red blood cells, white blood cells and platelets.
The counter was designed for measuring white blood cells (WBC), red blood cells (RBC), and hemoglobin content (HGB), hematocrit (HCT); mean (red) cell volume (MCV), mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC) and platelets (PLT), Neutrophil (NEUT), Lymphocytes (LYMPH) and Monocytes (MONO). The WBCs were analyzed by the flow cytometry method using semiconductor laser and the RBCs count was done by the RBC detector using the Hydro Dynamic Focusing method (DC Detection). HGB was analyzed by the HGB detector based on the SLS hemoglobin detection method and HCT by the RBC cumulative pulse height detection method. The MCHC was calculated with RBC, HGB and PLT by the Hydro Dynamic Focusing method (DC Detection) or flow cytometry method using semiconductor laser. As the measurements were done, the blood was continually shaken on a shaking device at room temperature.
Statistical analysis was accomplished by using a paired test to analyze the mean and standard deviations of different experimental groups. The null hypothesis was for no statistical difference between the means of the different groups. (H0: M1=M2=M3=M4 where M1, M2, M3, M4 are the mean of the experimental groups). A significant difference was accepted between the means when the p value was less than 5 % (P< 0.05)
BLOOD SMEAR PREPARATION
In preparation of blood smear two samples were taken, one from female (31 year, normal) and another from male (38 year old, normal). Erythroctes morphologiocal abnormalities were classified as echinocytes (crenated cells), dacrocytes (tear drop cells), stomatocytes, keratocytes (bite cells) and schistocytes (fragmented cells) cells. The blood smear were prepared in Institut Perubatan dan Pergigian Termaju (Advanced Medical & Dental Institute),Universiti Sains Malaysia.
The slides were cleaned with 70% alcohol and left to air dry. In preparation of the blood smear, a small drop of well-mixed blood was placed in the centre line of the slide about 1cm from one end. Immediately a spreader (a glass microscope slide with at least one smooth end, broken off at one corner of this end using a glass cutter, leaving width of c15 mm) was placed in front of the drop at an angle of 30ºC to the slide and moved backward to make contact with drop of blood. The drop of bread was spread along the slide making sure the spreader was not lifted up until the last trace of blood was spread out. The film was left to air dry as the patient’s identification was entered at the edge of the film.
PLATE 1: PREPARING SLIDES ON A SLIDE
Immediately after air drying, the films were fixed to preserve the morphology of the blood cells. The films were placed in a tray containing alcohol (methanol) for 2-3 minutes. Then, Romanowsky staining was done using the Wright’s method. The main components of the stain is a cationic (basic) dye which binds to anionic sites and gives a blue-grey color to nucleic sites, nucleoproteins, basophils granules and weakly to neutrophils granules for example azure B.
The other major component of this stain is an anionic (acidic) dye which binds cationic sites on proteins and gives an orange-red color to hemoglobin and eosinophils for instance eosin Y. The pH was carefully controlled through the use of a phosphate buffer at pH of 6.8. In low pH, the stain will take on a pinkish tint, and nuclear structures will be poorly stained whereas in high pH, all intracellular structures such as nuclei will be blue-black in color with poorly defined structure. Blood Smear preparation continuation with numbering
CHAPTER IV: RESULTS
The results of the blood smear after the green laser irradiation at different time periods for the male () and female () are shown below in Table 4.1 and Table 4.2 respectively.
Table 4.1: Observed effects of irradiation of six types of poikilocytes from male sample Type of poikilocyte Time of green laser irradiation Zero (Sec) Control One (Sec) Three (Sec) Six (Sec) Ten (Sec)
Echinocytes (“crenated cells”) 0 0 0 0 0 Dacrocytes (“tear drop cells”) +- 0 0 0 0 Stomatocytes +- +- 0 +- +- Keratocytes (bite cells) 0 +- +- 0 0 schistocytes (“fragmented cells”) 0 0 0 +- 0 target cells 0 0 0 0 0
Table 2.1: Observed effects of irradiation of six types of poikilocytes from female Type of poikilocyte Time of green laser irradiation Zero (Sec) Control One (Sec) Three (Sec) Six (Sec) Ten (Sec) Echinocytes (“crenated cells”) ± 0 0 0 0 Dacrocytes (“tear drop cells”) ± 0 ± 0 0 Stomatocytes + ± ± ± ± Keratocytes (bite cells) ± 0 0 0 0 schistocytes (“fragmented cells”) ± 0 0 0 0 target cells 0 ± 0 0 0
The results of the paired test to analyze the mean and standard deviations of different experimental groups. research showed the effect of low level laser light on the rheology of different blood cells as well as a change in the number of cells as below:
RED BLOOD CELLS:
Observations of green laser irradiation (532nm) on red blood cells (RBC) at different energy effluence in different time periods Time periods (in second) Irradiation (532nm, J/cm2) Mean p value n = 25 Control Irradiated 1 0.5 4.36 ± 0.92 4.46 ± 0.94 <0.05 3 1.5 4.11 ± 0.69 4.11 ± 0.91 > 0.05 6 3.0 4.31 ± 0.74 4.39 ± 0.87 > 0.05 10 5.0 4.41 ± 0.59 4.45 ± 0.63 > 0.05 n=number of samples In the paired sample test for the four experimental groups in the study, 3, 5 j/cm2 irradiation group showed a significant increase in the red blood cells of male patients (p<0.05). 0.5j/cm2 irradiation group showed p= 0.00 Hemoglobin: 3, 5j/cm2 irradiation group showed an increase in hemoglobin. Time periods (in second) Irradiation (532nm, J/cm2) Mean p value n = 25 Control Irradiated 1 0.5 12.70 ± 2.49 13.54± 2.37 >0.05 3 1.5 11.87± 2.67 11.94± 2.44 >0.05 6 3.0 12.14± 1.97 12.28± 2.25 > 0.05 10 5.0 12.42± 2.22 12.50± 2.22 > 0.05 n=number of samples 0.5j/cm2 irradiation group showed p=0.00 MCHC Time periods (in second) Irradiation (532nm, J/cm2) Mean Control Irradiated p value 1 0.5 33.70± 1.04 34.07± 0.95 <0.05 3 1.5 33.86± 0.96 34.09± 1.03 <0.05 6 3.0 33.29± 1.47 33.34± 1.71 > 0.05 10 5.0 33.17± 1.73 33.54± 1.91 < 0.05 n=number of samples
MCV Time periods (in second) Irradiation (532nm, J/cm2) Mean p value n = 25 Control Irradiated 1 0.5 85.94± 3.86 85.22± 3.90 <0.05 3 1.5 86.55± 5.95 85.67± 5.87 <0.05 6 3.0 84.86± 5.31 84.28± 5.37 < 0.05 10 5.0 84.62± 9.96 83.78± 9.78 < 0.05 n=number of samples MPV Time periods (in second) Irradiation (532nm, J/cm2) Mean p value n = 25 Control Irradiated 1 0.5 10.02± 0.82 10.34± 0.87 <0.05 3 1.5 10.28± 1.07 10.49± 1.18 <0.05 6 3.0 9.87± 0.82 10.11± 0.83 < 0.05 10 5.0 9.86± 0.77 9.92± 0.54 > 0.05 n=number of samples
PLT Time periods (in second) Irradiation (532nm, J/cm2) Mean p value n = 25 Control Irradiated 1 0.5 256.16±148.33 254.04± 137.59 >0.05 3 1.5 186.32± 95.09 217.72± 106.83 <0.05 6 3.0 252.28±104.97 246.96±109.95 > 0.05 10 5.0 222.96±101.61 234.52± 120.46 > 0.05 n=number of samples PDW Time periods (in second) Irradiation (532nm, J/cm2) Mean p value n = 25 Control Irradiated 1 0.5 12.080±1.92814 11.86± 1.51 >0.05 3 1.5 12.25± 2.07 12.28± 2.54 >0.05 6 3.0 11.38±1.68 11.40±1.64 > 0.05 10 5.0 11.38±1.49 11.23± 1.37 > 0.05 n=number of samples
RDW-CV Time periods (in second) Irradiation (532nm, J/cm2) Mean p value n = 25 Control Irradiated 1 0.5 14.70±1.93 14.57± 1.93 >0.05 3 1.5 14.78± 1.53 14.64± 1.46 >0.05 6 3.0 15.10±2.32 15.08±2.34 > 0.05 10 5.0 15.02±1.70 14.98± 1.72 < 0.05 n=number of samples RDW-SD Time periods (in second) Irradiation (532nm, J/cm2) Mean p value (n = 25 Control Irradiated 1 0.5 44.9±4.94 44.50± 4.81 >0.05 3 1.5 46.02± 6.01 45.48± 5.95 >0.05 6 3.0 45.96±7.50 45.7160±7.39835 < 0.05 10 5.0 44.56±4.93 44.40± 4.80 > 0.05 n=number of samples
Only 0.5j/cm2 irradiation group showed a significant increase in hematocrit (p<0.05) Time periods (in second) Irradiation (532nm, J/cm2) Mean p value n = 25 Control Irradiated 1 0.5 12.7040 ± 2.49090 13.5440± 2.37207 >0.05 3 1.5 11.8716± 2.66635 11.9360± 2.43856 >0.05 6 3.0 12.1360± 1.96996 12.2800± 2.25000 > 0.05 10 5.0 12.4200± 2.22299 12.5040± 2.21726 > 0.05 n=number of samples
Thus it is evident from the above results at very low effluence of 0.5j /cm2, only a change in hematocrit is possible while 3 and 5j /cm2 increase the red blood cells and hemoglobin.
The test results showed the following changes in irradiated groups:
The increase in white blood cells and red blood cells seen with 3 and 5 j /cm2 was two times the increase seen with 0.5 and 1.5j/cm2 groups. Similarly HGB increased with 0.5, 3 and 5j /cm2 but decreased two times in 1.5j /cm2 group and the same change was seen in non-irradiation group. HCT increased to double in 0.5 and 5j /cm2 group where decreased in 3j /cm2 group and even more in 1.5j /cm2 group. Neutrophils increased to double in 0.5, 3 and 5j /cm2 group but decreased in 1.5j /cm2 group. Lymphocytes increased in 0.5, 3 and 5j /cm2 group but double increased in 1.5j /cm2 group. MCV and MCHC increased to double in all the groups. Platelets double decreased in all groups except in 5j /cm2 in which no change was observed.
These results show a positive effect of 5j /cm2 effluence power on almost all the cells under investigation, whereas the other three intensities show a variation their effects on different indices. Irradiation groups with different laser effluence showed the following results with gender differences: In males: In 0.5 j/cm2 group HGB, RBC’s and HCT increased significantly In 1.5 j/cm2 group HGB, RBC’s and HCT increased significantly In 3j/cm2 group HGB and RBC’s increased significantly In 5j/cm2 group HGB and RBC’s increased significantly. In females: In 0.5 j/cm2 group RBC’s and HGB increased significantly whereas HCT decreased non-significantly. In 1.5, 3, 5 j/cm2 groups RBC’s, HGB and HCT decreased non-significantly.
When the above information was extracted from the results on gender basis, it became evident that some gender difference is also an important factor in determining the efficacy of low level laser therapy on blood.
CHAPTER V: DISCUSSION
The main objective of this study is to explore the bio stimulatory effect of low level laser on human blood samples. We conducted this research to determine the effect of low-level laser therapy (LLLT) on some rheological properties of human blood in vitro by using laser pointer 532 nm, maximum output 100mw.It also aims to evaluate the effect of this therapy on reducing inflammation be demonstrating the transformations of blood cells, the effect of LLLT dose response of blood cell and the changes in blood cell counts. Laser therapy is applied on body tissues which may be cells or culture to bring a change in tissue functions and properties. More than 130 double blinded studies have confirmed the therapeutic benefit of low level laser therapy. Laser therapy is a matter of dose and treatment technology as it is with any other therapy. The power output of laser is important especially for dose calculation. The depth of penetration is dependent on wavelength of light.
The objective of our research was to determine the effects of green laser light on the rheology of different blood cells, in vitro. We evaluated the counts of red and white blood cells, HGB, HCT, MCV, MCHC, PLT and neutrophils.
We demonstrated many beneficial effects of green laser light irradiation on erytherocytatory, leucocytatory, aggregability indices. The bio stimulatory effect of Low level laser therapy on red cells was seen with changes in cell membranes, thus increasing the red cell functionality.The physically and chemically stressed erythrocyte membranes can be revitalized and brought back to functionality for performing its oxophoric function in transfusion reactions.
From the results of our research, we can say that low level laser therapy affects various rheological properties of different blood cells for example red cell deformability, aggregation of cells, critical stress on the cells during preservation time, leucocytarory, erytherocytatory indices, ESR etc. The best effluence power that has a positive effect on almost all blood cells and indices is 5j /cm2. It increased white blood cells, red blood cells, hemoglobin with a non-significant decrease in hematocrit. Thus from our research it is proved that low level laser therapy with diode laser 532nm and a high power of 100 Mw is advantageous for revitalizing the functional capability of preserved blood and also increases the number of blood cells, thus increasing the function of this blood when injected to recipient.
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