ex 3 report
TRANSCRIPT
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Permeability properties II:
Mammalian Erythrocytes
Diamos, Pineda, Rañola, Saballo and Tancio
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Schematic Diagram
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Prescribed NaCl Concentrations
0.15M
0.13M
0.12M
0.11M
0.10M
0.08M
0.06M
0.04M
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Methodology
Acquire blood sample from a
volunteer
Prepare prescribed concentrations of
NaCl solutions
Turn on and warm up the
spectrophotometer
Suspend 0.10mL blood in 5mL 0.15M
NaCl
Suspend 0.10mL blood in 5mL distilled H2O.
Set wavelength to 620nm
Cover and invert to mix settled blood
with solution
Measure absorbance and transmittance
Repeat steps 6-8 for the other
concentrations
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Results
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Table 1. Salt concentration vs. absorbance and transmittance
Concentration of NaCl (M) Absorbance Transmittance (%)
0.04 -0.076 115.8
0.06 1.383 4.1
0.08 0.823 15.0
0.10 1.397 9.0
0.11 1.412 3.9
0.12 1.395 4.0
0.13 1.460 3.5
0.15 1.509 3.1
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Figure 1. Salt concentration (M) as a function of absorbance (nm)
0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16
-0.2
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
NaCl Concentration (M)
Ab
sorb
an
ce (
nm
)
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Figure 2. Salt concentration as a function of transmittance
0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.160
20
40
60
80
100
120
140
NaCl Concentration (M)
Tra
nsm
itta
nce (
%)
Study Question #2: Explain the appearance of the curve on the graph showing absorbance (or transmittance) values versus NaCl concentration).
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Why oh why?
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RBC Ghost
Low NaCl concentration=hemolysis=less intact cells=less absorbance=higher transmittance
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Study Questions1. Do all RBCs in any
one solution hemolyze at the same time? Explain.
No, they don’t, because at any one solution, the cells and the ions would not interact simultaneously, thus, hemolysis will not occur at the same time for all erythrocytes.
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3. What is the importance of the absorbance values in relation to the permeability in the erythrocyte?
More permeable=greater movement of water and solutes into and out of RBC=more prone to lysing and crenation=less absorbance values
Less absorbance value, more permeable!
Study Questions
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4. Predict what would happen to the absorbance values obtained if you used saline solutions at a concentration greater than 0.15M?
- Normal physiological saline concentration- O.15 M or 9% NaCl - Hypertonic - All erythrocytes present in the solution would shrink (crenation), releasing all contents from the cells less intact cells less absorbance values
Study Questions
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Figure 3. Theoretical graph of absorbance at increasing NaCl concentrations
0.04 0.06 0.08 0.1 0.11 0.12 0.13 0.15
-0.2
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
NaCl Concentration (M)
Ab
sorb
an
ce (
nm
)
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Figure 4. Theoretical graph of absorbance at increasing NaCl concentrations
0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 0.2 0.220
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
NaCl Concentration (M)
Ab
sorb
an
ce (
nm
)
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Table 2. Salt concentration vs. absorbance and transmittance (Grp 1 -
female)
Concentration of NaCl (M) Absorbance Transmittance (%)
0.04 0.019 95.6
0.06 0.286 5.2
0.08 0.634 23.2
0.10 1.265 5.5
0.11 1.353 4.5
0.12 1.426 3.8
0.13 1.394 4.1
0.15 1.447 3.6
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0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.160
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
NaCl Concentration (M)
Ab
sorb
an
ce (
nm
)
Figure 5. Salt concentration (M) as a function of absorbance (nm)
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0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.160
20
40
60
80
100
120
NaCl Concentration (M)
Tra
nsm
itta
nce (
%)
Figure 6. Salt concentration as a function of transmittance
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Table 3. Salt concentration vs. absorbance and transmittance (Grp 3 –
female with fever)
Concentration of NaCl (M) Absorbance Transmittance (%)
0.04 0.044 90.4
0.06 1.430 3.7
0.08 1.224 6.0
0.10 1.459 3.5
0.11 1.435 3.7
0.12 1.682 2.1
0.13 1.491 3.2
0.15 1.537 2.9
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Figure 7. Salt concentration (M) as a function of absorbance (nm)
0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.160
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
NaCl Concentration (M)
Ab
so
rba
nce
(n
m)
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Figure 8. Salt concentration as a function of transmittance
0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.160
10
20
30
40
50
60
70
80
90
100
NaCl Concentration (M)
Tra
nsm
itta
nce
(%
)
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5. What is the fragility test? How is this test clinically applied?
This is a test to determine if the RBCs are more likely to break down. It is done by subjecting the RBCs in decreasing strengths of hypotonic saline solutions. This is clinically applied in evaluating hemolytic anemia, especially hereditary spherocytosis, and in evaluating hemolytic states.
Study Questions
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Blood is drawn from a vein In the laboratory, Red blood cells are tested with a solution that makes them swell, in order to determine how fragile they are.
Normal RBCs – initial hemolysis at 0.45 – 0.39% salt concentrations
- complete hemolysis at 0.33 – 0.30% salt concentration
Increased fragility -> hereditary spherocytosis Reduced fragility -> thalassaemia, sickle cell
anemia, obstructive jaundice
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Typical Graphs for RBC Osmotic Fragility
An increase in osmotic fragility is caused by decreasing the surface area of the cell (such as in a spherocyte).
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References
Franklin Institute. 2004. Red Blood Cells: Riding on the Road. The Franklin Institute: The Human Heart. Retrieved November 24, 2012 from http://www.fi.edu/learn/heart/blood/red.html.
Liang, B. 2010. Red Blood Cells. Wisc-Online. Retrieved November 24, 2012 from http://www.wisc-online.com/objects/ViewObject.aspx?ID=ap14604. Fall. 2009. Red Blood Cell Membrane Permeability. Home.Zhaw.Ch. Retrieved November 24, 2012 from https://home.zhaw.ch/~fusa/PSS_VLE/PSS_CASE_04/ACTIVITIES/INVESTIGATIONS/EXPERIMENTS/Exp_11/Files/Blood_Cells_Permeability.pdf
Von Blum, R. 2003. Experimental Studies of Permeability in Red Blood Cells. Ableweb.org. Retrieved November 24, 2012 from http://www.ableweb.org/volumes/vol-2/5-von_blum.pdf.