Hemoglobin:- Part 2 – Hemoglobin Electrophoresis, (Hb electrophoresis)
- Hemolysate is prepared from blood in EDTA, citrate, or heparin.
- Use fresh or refrigerate the sample.
Methods to prepare the hemolysates:
- Method 1: Take blood clotted or RBCs with an equal volume of water.
- Keep overnight in the freezer (or freeze overnight).
- The supernatant will be ready.
- Method 2: 1 gram ethylenediaminetetracetic acid (EDTA).
- 0.1 gram of saponin.
- Dissolve in 500 ml of D. water.
- Take normal saline washed Red blood cells.
- Mix an equal volume of reagent and the washed RBCs for 5 minutes.
- Hemolysate will be ready.
- Method 3: Preparation of hemolysate:
- Centrifuge the EDTA blood and remove the upper liquid portion (supernatant plasma and buffy coat).
- Wash these cells with saline three times, and discard the saline from the last centrifuge.
- Add an equal volume of distle water and 1/2 volume of carbon tetrachloride.
- Now shake it vigorously.
- Centrifuge for 5 minutes at 1500 G.
- Three layers will form.
- Upper hemolysate.
- Middle RBC leftover (stroma).
- Carbon tetrachloride (CCl4).
- Carefully pipette off the upper layer of the hemolysate (supernatant). This will look ruby color and will be clear.
- Adjust the hemoglobin concentration with distle water 70 to 100 g/L.
- This is ready to run the hemoglobin electrophoresis.
- If you want to store for another time, add one drop of potassium cyanide (KCN = M/100); otherwise, you can run electrophoresis immediately.
Principle of Hemoglobin Electrophoresis
- The first hemolysate from the EDTA blood is made, and then it is run for electrophoresis, where Hb is separated into different bands.
- We can find the presence of an abnormal Hb pattern.
- Cellulose acetate or starch gel electrophoresis is run on the hemolysate at a pH of 8.6.
- Then Hb is quantified by elution and spectrophotography.
- Or by a densitometer.
- HbF is alkali resistant, so it needs to quantify by another method.
Purpose of the test (Indications)
- This will detect abnormal hemoglobinopathies and quantify them.
- This Hb electrophoresis will diagnose :
- Sickle cell anemia.
- Other abnormal hemoglobins.
Precaution for Hemoglobin Electrophoresis
- Blood transfusion in the last 12 weeks may alter the electrophoretic pattern.
The principle of Electrophoresis
- It is a migration of charged solutes or particles in a liquid medium under the electric field’s influence.
- Positive ions (cations) moves towards the cathode.
- Negative ions (anions) moves towards the anode.
Pathophysiology of Hemoglobin electrophoresis
- There are various types of hemoglobins, like A1, A2, S, C, and F.
- The hemoglobin moves at different rates in the electromagnetic field and forms various bands.
- The patient sample is compared to the normal Hb pattern.
- Each band is quantitated as a percentage of total hemoglobin.
- Hb A1 is the major hemoglobin in the normal RBC.
- While Hb A2 is the minor component (2% to 3 %).
- Hb F is the main hemoglobin in the fetal RBC. There is a minimal amount in the normal adult.
- Hb F, more than 3% after 3 years, is considered abnormal.
- Hb F can carry O2 when a small amount is present in the blood, like in the fetus.
- Hb S and C occur in American blacks.
- RBCs with Hb C have decreased lifespan and are easily lysed.
Normal values of hemoglobin
- Hb A1 = > 95 % (95 to 98 %).
- Hb A2 = 1.5 to 3.7 %
- Hb F = < 2 % (0.8 to 2 %).
- Hb S = 0 %.
- Hb C = 0 %.
- Newborn Hb F = 50 to 80 %.
- <6 months = <8 %.
- >6 months = 1 to 2 %.
Hb electrophoresis pattern in different conditions:
- Hemoglobin electrophoresis produces a good separation of Hb S from Hb A and C.
- In sickle cell anemia, cord blood electrophoresis show Hb F and Hb FS pattern, where Hb F consists of 60% to 80% of the total.
- After the age of 3 to 6 months, electrophoresis shows an SS pattern (80% to 90% Hb S), remaining is Hb F.
- Sickle cell trait has more than 50% of Hb A than Hb S. Therefore more Hb A than Hb S.
- S-Beta-Thalassemia has 50% or more Hb S with about 25% Hb A and more Hb A than Hb F.
- Hb A and Hb F can not be separated in some cases reliably.
- Hb C and Hb A2 migrate separately on citrate, where these will migrate together on cellulose acetate.
- Citrate agar gives a little better separation of Hb F from Hb A in newborn cord blood.
- Hb F can be identified and quantitated by the alkali denaturation method or by suitable electrophoresis.
The pattern of Hb electrophoresis in Thalassemia:
|Hemoglobin||Normal||Thalassemia Major||Thalassemia Minor|
|Hb F||< 1%||10 to 98%||Variable|
|Hb A||97%||Absent||80 to 90%|
|Hb A2||1 to 3%||Variable||5 to 10%|
In Major Thalassemia = Hb F = 98%, HbF = 2%
The pattern of electrophoresis:
|A > S||Sickle cell trait, sickle alpha-thalassemia|
|S, F, and no A||Sickle cell anemia, Sickle-beta thalassemia|
|S > A and F||Sickle – beta-thalassemia|
|A > C||Hb C trait|
|C, F, and no A||Hb C disease, HbC- beta-thalassemia|
|C > A||Hb C – beta-thalassemia|
Hemoglobinopathy pattern under different conditions:
|Disease||Hb A1||Hb A2||Hb F||Hb S||Hb C||Hb H|
|Sickle cell anemia||0||2% to 3 %||2 %||95 to 98 %||0||0|
|Sickle cell trait||50 to 60 %||2 to 3 %||2 %||35 to 45 %||0||0|
|β-thalassemia major||0||0 to 15 %||85 to 100 %||0||0||0|
|β-thalassemia minor||50 to 85 %||4 to 8 %||1 to 5 %||0||0||0|
|Hb C disease||0||2 to 3 %||2 %||0||90 to 100 %||0|
|Third. Generation alpha thalassemia.||65 to 90%||0||0||0||0||5 to 30 %|