Diamos, Pineda, Raola, Saballo and Tancio

Schematic Diagram

Prescribed NaCl Concentrations

0.15M
0.13M 0.12M 0.11M

0.10M
0.08M 0.06M 0.04M

Methodology
Prepare prescribed concentrations of NaCl solutions Acquire blood sample from a volunteer 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

Concentration of NaCl (M) 0.04 0.06 0.08

Absorbance
-0.076 1.383 0.823

Transmittance (%)

115.8
4.1 15.0 9.0 3.9 4.0 3.5 3.1

0.10
0.11 0.12 0.13

1.397
1.412 1.395 1.460

0.15

1.509

1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 0 -0.2 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16

Absorbance (nm)

NaCl Concentration (M)

Figure 1. Salt concentration (M) as a function of absorbance (nm)

140 120 100

Transmittance (%)

80 60 40 20 0 0 -20 0.02 0.04 0.06 0.08

Study Question #2: Explain the appearance of the curve on the graph showing absorbance (or transmittance) values versus NaCl concentration).

0.1

0.12

0.14

0.16

NaCl Concentration (M)

Figure 2. Salt concentration as a function of transmittance

RBC Ghost

Low NaCl concentration=hemolysis=less intact cells=less absorbance=higher transmittance

Study Questions

Study Questions
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
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

1.6

1.4

1.2

Absorbance (nm)

0.8

0.6

0.4

0.2

0.04
-0.2

0.06

0.08

0.1

0.11

0.12

0.13

0.15

NaCl Concentration (M)

Figure 3. Theoretical graph of absorbance at increasing NaCl concentrations

1.8 1.6 1.4

Absorbance (nm)

1.2 1 0.8 0.6 0.4 0.2 0

0.05

0.1

0.15

0.2

0.25

NaCl Concentration (M)

Figure 4. Theoretical graph of absorbance at increasing NaCl concentrations

Table 2. Salt concentration vs. absorbance and transmittance (Grp 1 female)

Concentration of NaCl (M) 0.04 0.06 Absorbance 0.019 0.286 Transmittance (%) 95.6 5.2 23.2 5.5 4.5 3.8 4.1

0.08
0.10 0.11 0.12 0.13 0.15

0.634
1.265 1.353 1.426 1.394 1.447

3.6

1.6

1.4

1.2

Absorbance (nm)

0.8

0.6

0.4

0.2

0 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16

NaCl Concentration (M)

Figure 5. Salt concentration (M) as a function of absorbance (nm)

120

100

Transmittance (%)

80

60

40

20

0 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16

NaCl Concentration (M)

Figure 6. Salt concentration as a function of transmittance

Table 3. Salt concentration vs. absorbance and transmittance (Grp 3 female with fever)
Concentration of NaCl (M)
0.04 0.06 0.08 0.10 0.11 Absorbance 0.044 1.430 1.224 1.459 1.435 Transmittance (%) 90.4 3.7 6.0 3.5 3.7 2.1 3.2 2.9

0.12
0.13 0.15

1.682
1.491 1.537

1.8 1.6 1.4

Absorbance (nm)

1.2 1 0.8 0.6 0.4

0.2
0 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16

NaCl Concentration (M)

Figure 7. Salt concentration (M) as a function of absorbance (nm)

100

80

Transmittance (%)

60

40

20

0 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16

-20

NaCl Concentration (M)

Figure 8. Salt concentration as a function of transmittance

Study Questions
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.

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

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).

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.wisconline.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/IN VESTIGATIONS/EXPERIMENTS/Exp_11/Files/Blood_Cells_Permeab ility.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.