Initially a pilot study was conducted. The experiment required four milliliters (ml) of blood from Wistar rat, 1
ml for each LED pen and 1 ml for the control, withdrawn by cardiac puncture with heparinized syringe. The
cardiac puncture was followed by the steps, in the precisely order, below:
• It was deposited 1 ml of blood in a eppendorf by the wall and this sample was irradiated for 10 minutes in a
distance of 5 cm from the surface of blood to the pen tip of the LED;
• The blood was removed with a volumetric pipette 100 microliters (μl) of irradiated blood and it was placed
in 6 test tubes containing 5 ml clean and dry hypotonic different concentrations of NaCl + 0.9% distilled water
[5]. The six concentrations contained the following values: Tube 1 = 0.7 ml 0.9% NaCl + 4.3 ml distilled
water, tube 2 = 1.4 ml 0.9% NaCl + 3.6 ml distilled water, tube 3 = 2 ml NaCl 3% + 0.9 ml distilled water,
tube 4 = 2.7 ml 0.9% NaCl + 2.3 ml distilled water, tube 5 = 3.4 ml 0.9% NaCl + 1.6 ml water distilled and
tube 6 = 4 ml 0.9% NaCl + 1 ml distilled water [5].
• The tubes were capped and mixed thoroughly, left to rest at room temperature for 30 minutes and homogenized
gently every 15 minutes;
• The samples were centrifuged at 1500 rpm (revolutions per minute) for 5 minutes;
• The supernatants were therefore isolated and removed with a pipette volume of 1000 μl and arranged in
buckets for analysis;
• It was analyzed the optical density (OD) of hemoglobin for each concentration of NaCl in a spectrophotometer
at 540 nm (specific value for hemoglobin analysis);
• The optical density of each supernatant with the optical density of the solution with NaCl [12%] was compared.
The supernatant of this solution is considered white space for preparation, because it has no hemolysis.
In trend curve according to the fragility, three intervals were determined: 1) the range between 0.12% and
0.36% NaCl; 2) the range between 0.36% and 0.60% NaCl and 3) the range between 0.60% and 0.90% NaCl
solution [5].
• The animals were sacrificed in CO2 chamber after blood collections.
By confirming the enforceability of the study, there were made 4 new collections that extrictly followed the
procedures mentioned above.
Initially a pilot study was conducted. The experiment required four milliliters (ml) of blood from Wistar rat, 1
ml for each LED pen and 1 ml for the control, withdrawn by cardiac puncture with heparinized syringe. The
cardiac puncture was followed by the steps, in the precisely order, below:
• It was deposited 1 ml of blood in a eppendorf by the wall and this sample was irradiated for 10 minutes in a
distance of 5 cm from the surface of blood to the pen tip of the LED;
• The blood was removed with a volumetric pipette 100 microliters (μl) of irradiated blood and it was placed
in 6 test tubes containing 5 ml clean and dry hypotonic different concentrations of NaCl + 0.9% distilled water
[5]. The six concentrations contained the following values: Tube 1 = 0.7 ml 0.9% NaCl + 4.3 ml distilled
water, tube 2 = 1.4 ml 0.9% NaCl + 3.6 ml distilled water, tube 3 = 2 ml NaCl 3% + 0.9 ml distilled water,
tube 4 = 2.7 ml 0.9% NaCl + 2.3 ml distilled water, tube 5 = 3.4 ml 0.9% NaCl + 1.6 ml water distilled and
tube 6 = 4 ml 0.9% NaCl + 1 ml distilled water [5].
• The tubes were capped and mixed thoroughly, left to rest at room temperature for 30 minutes and homogenized
gently every 15 minutes;
• The samples were centrifuged at 1500 rpm (revolutions per minute) for 5 minutes;
• The supernatants were therefore isolated and removed with a pipette volume of 1000 μl and arranged in
buckets for analysis;
• It was analyzed the optical density (OD) of hemoglobin for each concentration of NaCl in a spectrophotometer
at 540 nm (specific value for hemoglobin analysis);
• The optical density of each supernatant with the optical density of the solution with NaCl [12%] was compared.
The supernatant of this solution is considered white space for preparation, because it has no hemolysis.
In trend curve according to the fragility, three intervals were determined: 1) the range between 0.12% and
0.36% NaCl; 2) the range between 0.36% and 0.60% NaCl and 3) the range between 0.60% and 0.90% NaCl
solution [5].
• The animals were sacrificed in CO2 chamber after blood collections.
By confirming the enforceability of the study, there were made 4 new collections that extrictly followed the
procedures mentioned above.
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