62 Kleihauer-Betke Test (Modified) (using a “Simmler” kit)

Principle

The Kleihauer-Betke test is an acid elution staining technique used to detect fetal red blood cells in the maternal circulation and to calculate the volume of fetal-maternal bleed (FMB). Fetal Hemoglobin (HbF) is resistant to acid elution. Adult Hemoglobin (HbA) is not resistant to acid elution. A thin blood smear is fixed, exposed to an acid buffer eluting solution and stained. Fetal rbc’s resist acid elution, retain their hemoglobin, and take up stain. Adult rbc’s lose their hemoglobin into the buffer so that only the stroma remains and they appear as ghost cells.

Specimen

Maternal blood sample should be collected as soon after delivery as possible. Cord blood is not acceptable for this procedure. The blood is anti-coagulated with EDTA. Store sample at 2°C-4°C until it can be assayed. The assay should be performed within 24 hours of collection.

Controls

A positive control may be prepared from a mixture of 1 part of cord blood and 9 parts of normal adult blood.

Reagents

1. Fetal cell fixing solution (80% reagent alcohol)-flammable
2. Fetal cell buffer solution (citrate buffer, 0.081 M)
3. Fetal cell stain (Erythrosin-B, Fast Green)

Procedure

1. Mix the blood well by gentle inversion.
2. Place 3 drops of normal saline (0.85%) and 2 drops of maternal 2-4% cell suspension into a labeled glass test tube. Place a cap on the tube and mix by gentle agitation.
3. Place 1 drop of diluted blood on a glass slide near one end. Prepare a thin film by drawing the edge of another slide through the drop of blood and across the slide.
4. Air dry the slide at room temperature. **Once dry, the slide should now be processed immediately throughout the entire procedure. The smear must be thin enough to be a “monolayer” of cells.
5. Place the smears for 5 minutes in a clean coplin jar containing sufficient fetal cell fixing solution to cover the smears.
6. Remove the smears and rinse thoroughly in distilled water. Allow the slides to drain dry.
7. Place the smears for 8-10 minutes in a clean coplin jar containing sufficient fetal cell buffer solution to cover the smears.
8. Remove the slides from the solution and immediately place in a clean coplin jar containing fetal cell stain for 3 minutes.
9. Remove slides from the fetal cell staining solution and rinse thoroughly in distilled water. Dry the slides at room temperature.
10. Slides should be examined dry under 10X or 40X.

Interpretation and Reporting

1. Fetal cells will stain a dark reddish-pink while adult cells will appear white to light pink with a darker center. Staining intensity of adult cells may vary slightly within lots of reagents, however, fetal and adult cells will be easily differentiated. Slides should be read within 24 hours.
2. This procedure is performed on Rh negative mothers who deliver Rh positive babies. If an Rh negative mother has an Rh negative baby there is no need for this procedure to be performed.
3. Check positive control smears to ensure the method worked.
4. Use the following method to calculate the actual FMB:
   1. Select area on smear where the cells are evenly distributed.
   2. Scan 8-10 fields under low power.
   3. If no fetal cells are detected, report as negative.
   4. If fetal cells are detected, count the number of fetal cells in 1000 cells (includes both adult and fetal cells) using either 10X or 40X.
   5. Record the number of fetal cells seen/1000 total cells.

Calculate the Fetal Cell Count (FCC) per 1000 and report as the Kleihauer-Betke (KB) KB i.e. 10 fetal cells seen in 1000 total cells= 10 fetal cells+990 adult cells

KB= number of fetal cells  =   10_=  0.01 in SI units

         number of adult cells     1000

0.01X100  is the calculation of the “volume” of the bleed. In this case a 1% bleed

Estimated maternal blood volume is 5000 mL

1.0/100 (0.01) X 5000 mL=50 mL of fetal blood circulating in mother

50 mL x 12µg = 600 µg of RhIg needed for this mother to prevent immunization.

Notes

1. Administration of RhIg therapy is dependent on the volume of fetal maternal bleed (FMB).
2. Some normal adult red blood cells may contain small amounts of Hgb F (0.020) and may stain slightly. They are termed intermediate cells and do not stain as distinctly as fetal cells. They are not of fetal origin. For the purpose of administering Rh immune globulin as little as 0.003 would warrant giving the patient Rh immune globulin.
3. The degree of elution of the adult hemoglobin may vary from patient to patient.
4. Some disease states may cause increased levels of Hgb F. These cells will stain just like fetal cells and no distinction can be made between adult and fetal cells (such as Sickle cell disease).