Hb electrophoresis (principle materials and procedure)

principle reagent material methods procedure risks and results

1. HB Electrophoresis Presented to: Miss.Mehreen Presented by: Shahid Hussain

2. Definition HB electrophoresis is used as a screening test to identify variant and abnormal hemoglobins HB electrophoresis help in diagnosis of diseases in which abnormal hemoglobin production occur Electrophoresis uses an electrical current to separate normal and abnormal types of HB in the blood Hemoglobin types have different electrical charges and move at different speeds. The amount of each hemoglobin type in the current is measured

3. Heamoglobin Abbreviated  Hb  or  Hgb  Iron containing oxygen transport metalloprotein  in the RBC  of almost all vertebrates as well as the tissues of some invertebrates Hemoglobin has an oxygen binding capacity of 1.34 mL O 2  per gram 1 molecule carry 4 molecule of O 2

4. Types Normal: HbA : 95%-98% HbA  2 : 1.5%-3.5% HbF: < 2% Abnormal Abnormal form of HB is also known as varient Abnormality in HB occur mostly due to genetic mutation Over 350 types of abnormal HB have been found HB C, D, E, M, and S are abnormal forms

5. Purpose A part of routine check up ( complete blood test) To diagnose blood disorder( thalassemia polycythemia rubra vera and sickle cell anemia ) To monitor treatment To screen for genetic condition Help couples find out how likely they are to have a child with certain forms of anemia that can be passed from a parent to a child (inherited)  when someone has had a positive Hemoglobin Solubility test

6. Principle It uses the principle of Gel electrophoresis. Different heamoglobin have different charges and according to those charges and the amount of heamoglobin, different chains move at different speed in gel and seperates.

7. Reagent Electrophoresis buffer(Tris/EDTA/borate (TEB), pH 8.5) Wetting agents (e.g, Zip Zone Prep solution) Fixative stain/solution(Ponceau S 5 g) Haemolysing reagents(0.5% Triton in 100 mg potassium cyanide) Destaining solution (3% acetic acid)

8. Materials Specimen: Blood Container:green-, or blue-top vacuum tube

9. Methods   Cellulose acetate (CA) electrophoresis Alkaline electrophoresis Citrate agar electrophoresis Alkaline and Citrate agar electrophoresis are the commonly used method

10. Procedure Sample collection(blood) Centrifuge samples at 1200  g  for 5 min Prepare the electrophoresis tank with TEB buffer Soak the cellulose acetate into buffer for 5 min Fill the sample well plate with 5 μl of each diluted sample and cover with glass slide to prevent evaporation Load a second sample well plate with Zip Zone Prep solution

11. Cont… Then Applying them to a blotter Blot the cellulose acetate strip twice between two layers of clean blotting paper Do not allow the cellulose acetate to dry Load the applicator by depressing the tips into the sample wells twice Place the cellulose acetate plates across the bridges After 25 min of electrophrosis immediately transfer the cellulose acetate to ponceau S and fix and stain for 5 min

12. Cont… Remove excess stain by washing for 5 min in the first acetic acid reservoir Label the membrane and store in protective envelope

13. Risks There is very little chance of a problem from having a blood sample taken from a vein Hematoma ( can lower the chance by keeping pressure on the site for several minutes ) Bleeding Infection at the puncture site Fainting or feeling lightheaded Swelling ( also called phlebitis. A warm compress can be used several times a day to treat this )

14. Result Results available after 1-2 days depending on lab If normal then no problem If abnormal then following conditions can occur depend on type of abnormal HB Higher than normal amounts of both HB A2 and F may mean a mild form of thalassemia is present High levels of HBF may be seen in a rare condition called hereditary persistence of fetal hemoglobin

15. Cont… HB S in high amounts means  sickle cell disease  HB C in high amounts means patients have anemia  and an enlarged spleen HB E in high amounts means patients have anemia and RBC size will be smaller then normal

16. What Affects The Test Reasons you may not be able to have the test or why the results may not be helpful include Having a blood transfusion in the past 3 months Having iron deficiency anemia This can cause falsely low results for hemoglobin A2

17. Applications Evaluation of unexplained hemolytic anemia Microcytic anemia unrelated to iron deficiency, chronic disease, or lead toxicity A peripheral smear with abnormal red cell features (eg, target cells or sickle cells) Positive family history of hemoglobinopathy Positive neonatal screen results Positive results on sickle cell or solubility test