Hemoglobin Electrophoresis

What is electrophoresis?

The movement of charged particles in a medium under an electric field

Why are acid and alkaline plates used for hemoglobin electrophoresis?

Alkaline plates identify the presence of potential irregular hemoglobin such as Hgb S and Hgb C while the acid plate confirms the presence of S or C and rules out other less common hemoglobin like D/G or E/O

Where is the point of application for acid and alkaline plates?

• alkaline - (x)CSFA

• acid - FAS(x)C

What is the point of application?

The point where the proteins are applied onto the plate, the origin of the electrophoresis

What are some factors affecting electrophoresis?

• pH (buffer)

• time

• temperature

• medium (gel)

• electric field

• charge, size, shape of the molecule

The pH of a buffer of a gel electrophoresis is 8.6. Protein A has a pI of 7.5 and protein B has a pI of 3.9. Which protein travels the farthest and why?

Protein B travels farther because the difference between the buffer pH and the molecule pI is greater.

What is the isoelectric point?

The point at which the positive and negative charge of a molecule is equal or the net is zero.

A positively charged molecule in electrophoresis moves towards the—

cathode

A negatively charged molecule in electrophoresis moves towards the—

anode

If an acid plate does not confirm the presence of Hgb S despite a band on the alkaline plate, this indicates—

the presence of Hgb D/G

If an acid plate does not confirm the presence of Hgb C despite a band on the alkaline plate, this indicates—

the presence of Hgb E/O

Name and explain the force that causes gamma globulins to migrate backwards towards the cathode instead of the anode despite the proteins being negatively charged

This is called electroosmosis and occurs when the force of the reaction is stronger than the force of the electrophoresis causing the gamma globulins to be swept backwards.

What are the common types of hemoglobin tested for in electrophoresis?

A, A2, F, S, C

What is the pH of an acid plate?

6.0

What is the pH of an alkaline plate?

8.6

What is the order of migration of an alkaline plate from top to bottom?

A, F, S, C/A2

What is the order of migration of an acid plate from top to bottom?

C, S, A, F/A2

On a plate with a pH of 8.6, a patient has over 25% of their hemoglobin in the final bottom band of the gel. What hemoglobinopathy could they have?

Irregular hemoglobin (Hgb C/E/O)

A patient presents with less than 5% of Hgb A, what can we assume they have?

Thalassemia

A patient comes in and a Hgb electrophoresis is performed. The patient has 80% Hgb S, 10% Hgb A2 and, 10% other. The next week, they come in and their Hgb electrophoresis shows 70% Hgb A, 10% Hgb S, 10% Hgb A2, and 10% other. How is this possible?

Transfusion, Sickle disease patients will never produce normal RBCs