You have been asked to estimate the content of hemoglobin in a test sample. You

Question

You have been asked to estimate the content of hemoglobin in a test sample. You decide to perform an assay where the concentration of hemoglobin in the test sample can be measured and compare with a hemoglobin standard curve whose range is across 25 to 800 ug/well. The test sample is treated identically (volume assayed is 10 ul) to the standard hemoglobin and gives an absorbance reading of 0.4. By using the kaleidagraph program to analyse the data, the following equation is produced: y = 0.0005 + 0.0018x Using the above information, answer the following questions:

a) what is the amount of hemoglobin in the test sample added to the well expressed in ug?

b) what is the concentration of the hemoglobin in the test sample expressed in g/L?

c) If the red blood cell count expressed as number of cells/L is 5.5 x 1012, what would be the amount of hemoglobin per red blood cell in the test sample? Express the results in picograms/cell.

Explanation / Answer

In the standard curve , hemoglobin amount in the range 25 to 800 ug is plotted against absorbance.

X axis represents the hemoglobin amount and Y axis represent the absorbance by each hemologobin amount.

y = 0.0005 + 0.0018x

Absorbance reading of test sample is 0.4 i.e value of y is 0.4. Put this value in the equation above

0.4 = 0.0005 + 0.0018x

0.4 -  0.0005 = 0.0018x

0.3995 / 0.0018 = x

x =221.9 ug

The amount of hemoglobin in the test sample is 221.9 ug

b) For determing the concentration divide the hemoglobin amount by voulme of test sample assay

concentration of test sample =  221.9 ug / 10 ul = 22.19 ug /ul

1 ug = 10-6 g and 1 ul = 10-6 L

22.19 X 10-6 g / 10-6 L = 22.19 g/L

Concentration of hemoglobin in test sample is 22.19 g/L

c) Number of red blood cells = 5.5 x 1012 cells / L

Test sample is 1 ul

1 L = 5.5 x 1012 cells

106 ul = 5.5 x 1012 cells (1 Litres = 106 ul (microlitres))

1 ul = 5.5 x 1012 / 106 cells

1 ul = 5.5 x 106 cells

10 ul = 5.5 x 106 x 10 cells = 5.5 x 107 cells

There are 5.5 x 107 red blood cells in 10 ul of test sample.

Amount of hemoglobin in test sample is 221.9 ug . Now we have to convert it to picograms

1 ug = 106 picograms

221.9 ug = 221.9 x 106 picograms

Amount of hemoglobin per red blood cells is = Amount of hemoglobin in test sample in picograms/ number of RBC in test sample

= 221.9 x 106 picograms / 5.5 x 107 cells

= 40.34 x 106-7 picograms/ cell

= 40.34 x 10-1 picograms/ cell

= 4.034 picograms per cell

amount of hemoglobin per red blood cell in the test sample is 4.034 picograms per cell

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