1. Practical Hematology Lab
Hemoglobin F Stain
Acid Elution (Kleihauer Betke Test)

2. Hemoglobin F Stain
The acid elution test is employed to assess the distribution of hemoglobin F in the red blood cell: This information is useful in helping to diagnose:
Hereditary persistence of fetal hemoglobin
In determining the presence of fetal red cells in the maternal circulation during pregnancy.

3. Reagents and Equipment
Fetal cell fixing solution is Ethyl alcohol, 80%.
Fetal cell buffer solution is Citric acid-phosphate buffer, pH 3.2 to 3.3.
Fetal cell stain:
1. Erythrosin B (eosin B) stain, 0.1% .
2. Ehrlich's acid hematoxylin.
Coplin jars.
Waterbath ( 37°C ).

4. Specimen
Make blood smears from venous blood collected in EDTA anticoagulant or from the fingertip (toe or heel).
For best result blood should be less than 6 hours old, although successful staining has been achieve on specimens refrigerated for up to 2 weeks
The smears should be fixed within 2 hours of preparation.

5. Principle
Blood smears are fixed with ethyl alcohol and then incubated in a citric acid-buffer solution In an acid medium (pH 3.2 to 3.3), hemoglobin F is resistant to elution from the red blood cell, while other types are removed from the red cells.
The slides are stained with hematoxylin (stains the white cell nuclei) and erythrosin B (stains the red cells).
The smears are then reviewed microscopically to mine the presence of hemoglobin F, and percentage of red blood cells containing fetal hemoglobin may be assessed.

6. Procedure
Prewarm citric acid-phosphate buffer. Place 50 mL of the buffer solution into a coplin jar and cover. Incubate at 37°C for 30 minutes. (Make certain the level of water in the incubator is level with, or above, the level of buffer in the coplin jar.)
Preparation of blood smears, Patient-Make several thin (a monolayer of cells) blood smears
Allow the blood smears to air-dry for at least 10 minutes.
Fix blood smears (patient and controls) in 80% ethyl alcohol for 5 minutes
Rinse the smears carefully in distilled water and allow to air-dry.

7. Procedure
Place the dry smears in the prewarmed citric acid-phosphate buffer solution for 5 minutes. At 1 and 3 minutes (of incubation), carefully lift each slide out of the buffer solution and immediately replace. This action will provide a gentle stirring of the solution.
After 5 minutes, remove the slides from the citric acid-phosphate buffer solution and carefully rinse with distilled water. Air-dry.
Stain the dry smears in acid hematoxylin for 3 minutes. Rinse with distilled water and remove as much of the water as possible from the smears by gently tapping one end of the slide on an absorbent material.

8. Procedure
Counterstain the smears with erythrosin B for 4 minutes. Rinse with distilled water, allow to air-dry, and cover slip (if desired).
Examine the slides microscopically, (oil immersion objective [1000]),

10. Procedure
To determine the percentage of red blood cells containing fetal hemoglobin:
Determine the average # of red cells/ field.
Number of hgb F RBC/field =
Number of Hb F RBC counted
Number of fields counted
% RBC with hgb F =
Number of HB F RBC/ field
× 100
Average number of RBC/field

11. Discussion
Reticulocytes may resist elution and would, therefore, give the appearance of cells containing hemoglobin F.
The degree of elution of adult hemoglobin may very from patient to patient.
Ethyl alcohol concentrations above 80% may cause the elution of hemoglobin F, while concentrations below 80% may cause morphologic alterations.

12. Discussion
In hereditary persistence of fetal hemoglobin, the amount of hemoglobin F in each cell is constant and, therefore, all of the red blood cells are consistently stained. Conversely, in diseases such as sickle cell anemia, thalassemia, acquired aplastic anemia, and several other hemoglobinopathies, the amount of hemoglobin F present in the red blood cells varies. This shows up as an inconsistent staining of the red cells.

13. Discussion
The pH of the citric acid-phosphate buffer is critical. A pH below 3.1 may cause elution of hemoglobin F from the red cells, while a pH above 3.3 may retard the elution of non-F hemoglobin from the cells.
A temperature above 25°C during fixation in the ethyl alcohol will inhibit elution of normal hemoglobin.