Electrophoresis & Kleihauer-Betke Test & Sickle Cells

Hemoglobinopathies

• Qualitative hemoglobinopathies: structural defects in the globin chain from amino acid substitution

• Quantitative hemoglobinopathies: reduced/absent synthesis of one type of globin chain

Hemoglobin Electrophoresis

• A method to separate and identify hemoglobin variants based on electrical charge & mobility. Used in newborn screening & prenatal diagnosis.

  • Sickle cell disease & trait

  • HbC disease & trait

  • Beta-thalassemia (HbF, HbA2)

  • Alpha-thalassemia (HbH, Bart’s)

• Two types of medium: cellulose acetate (alkaline) & citrate agar (acidic)

Normal Adult

• HbA: 95-98%

• HbA₂: 2-3%

• HbF: <1%

Cellulose Acetate Medium

• Hemoglobins are negatively charged that migrate toward anode

• Alkaline pH ~8.6

• Hb-S, Hb-D, HbG, Hb-Lepore co-migrate

• HbC, HbA_2,HbE & HbO-Arab, HbC-Harlem co-migrate

• Fastest to Slowest A → F → S/D → C/A₂

• Mnemonic A Fat Slow Crawling Animal

Acid Citrate Medium

• Hemoglobins are positively charged that migrate toward cathode

• Acidic pH ~6

• Distinguish HbS from HbD & HbG. Next, HbC from HbE/O/Arab

• Fastest to Slowest: HbF → HbA → HbS → HbC

• Mnemonic: Father And Son’s Cat

Sources of Error in Electrophoresis

Preanalytical

• Hemolyzed/clotted specimens

• Recent transfusion that will mixed RBC

Analytical

• Overloaded or underloaded samples

• Poor staining

Post analytical

• Faint bands

• Not considering clinical context

Sickle Cell Disease or Trait

• Sickle trait: HbA + HbS

• Sickle cell disease: predominantly HbS, some HbF, no HbA

Hemoglobin C Disease or SC Disease

• Hemoglobin C disease: predominantly HbC

• SC-Disease: Bands for HbS and HbC, no HbA

Beta-Thalassemia Major

Predominantly HbF, few HbA₂, absent HbA

Kleihauer-Betke Test {Formula}

• Detect and quantify fetal RBC in maternal blood.

• Assess fetomaternal hemorrhage (fetal RBC enter maternal circulation), help guide the Rh immune globulin dosage.

• The limitation is maternal may have thalassemia contains high HbF, and may overestimate fetal cells

• Acid elution (remove hemoglobin but retains membrane) of RBC, HbF is resistant to it so remains pink/red

• Observe 10 fields

• $$\%\text{fetal cells}=\frac{\text{fetal RBCs}}{\text{total RBC}}\cdot100$$

Kleihauer-Betke SOP

1. Use EDTA blood

2. Dilute blood by adding 3 drops 0.85% saline, 2 drops of blood or control(1 cord blood : 9 adult blood)

3. Make a thin blood smear with 1 drop of diluted blood, and air dry

4. Fix the smear with 80% reagent alcohol for 5 minutes

5. Rinse and dry

6. Acid elution with citrate buffer 0.081M for 10 minutes

7. Fetal cell stain erythrosin-B, stands for 3 minutes

8. Rinse and dry for 15 minutes

9. Read within 24 hours

Alpha-Thalassemia

HbH or HbBart’s (fastest and ahead of HbA) in alkaline electrophoresis

SickleScreen Hemoglobin Screening Principle

• HbS is not soluble when deoxygenated

• A surfactant is used to lyse RBC releasing hemoglobin

• Dithionite (sodium hydrosulfite) reduced hemoglobin, HbS becomes insoluble and precipitates. Normal hemoglobin remains soluble and clear

• Phosphate buffer maintains pH and promotes differential solubility

• Cannot distinguish sickle trait from disease

SickleScreen SOP

1. Use EDTA blood

2. Add 4mL of phosphate buffer

3. Add 50μL of whole blood and shake vigorously

4. Incubate for 10-20 minutes

Sodium Metabisulfite Sickling SOP

Utilize sodium metabisulfate to deoxygenate RBC. Does not distinguish sickle trait from disease. Positive may occurs for other abnormal hemoglobin (HbC, HbHarlem, HbBart’s). May be false negative if HbS < 10%

1. 1 drop of blood, 2 drops of sodium metabisulfite (1 if anemic) onto a slide

2. Seal the slide with coverslip rimmed with petroleum jelly

3. Incubate for 30 minutes

4. Examine the slides, in the absence of sickling, the slides should be re-examined in 24 hours