principle reagent material methods procedure risks and results

2. Presented to:
Miss.Mehreen
Presented by:
Shahid Hussain

3. 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

4. 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 O2 per gram
 1 molecule carry 4 molecule of O2

5. 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

6. 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

7. 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.

8. 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)

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

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

11. 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

12. 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

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

15. 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)

16. 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

17. 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

19. 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

20. 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