61 Fetal Bleed Screening Test

Principle

The fetal bleed screening test (“rosette test”) detects D positive red cells in the blood of a D negative woman whose fetus or recently delivered infant is D positive. When reagent anti-D is added to maternal blood containing the D positive fetal cells, fetal red cells become coated with anti-D. When D positive reagent cells from the manufacturer are subsequently added, easily visible rosettes are formed with several red cells clustered around each antibody-coated D positive cell. This is a “qualitative” method of detecting Rh positive fetal cells in maternal circulation.

Specimen

• A 2% to 4% saline suspension of washed red cells from a maternal blood sample.
• It is best to wait about an hour after delivery to allow any fetal blood to mix thoroughly in the maternal circulation, but the sample should be collected as soon as possible after that.

Controls

• Negative control: a 2% to 5% saline suspension of washed red cells known to be D antigen negative
• Positive control: a 2% to 5% saline suspension of a mixture containing approximately 0.6% D positive red cells and 99.4% D antigen negative red cells. This control can be prepared by adding 1 drop of a 2% to 5% suspension of D antigen positive control cells to 15 drops of a 2% to 5% suspension of washed D antigen negative control cells. Mix well, then add 1 drop of this suspension to 9 drops of the 2% to 5% suspension of D antigen negative red cells. Mix well.

Reagents

1. Indicator red cells: a 2% to 5% saline suspension of group O, R2R2 red cells (ficinized).
2. High protein reagent anti-D serum. Some monoclonal/polyclonal blended reagents are unsuitable for use in this method.

Procedure

1. Make a 2-4% saline suspension of the well-mixed maternal red blood cells to be tested in an appropriately labeled tube
2. Add one drop of the anti-D serum to a properly labeled test tube.
3. Place 1 drop of the maternal red blood cell suspension in the labeled tube.
4. Mix well and incubate for 15 minutes at 37°C.
5. Wash the red blood cells six times with saline (use 4X and 2X on cell washer).
6. Decant the saline completely after the last wash.
7. Add one drop of indicator cells and mix well by gently shaking the tube.
8. Centrifuge immediately for 15 seconds.
9. Re-suspend the red blood cell button completely and examine for mixed field agglutination then use the inverted microscope to confirm presence of “rosettes”.

Interpretation and Reporting

1. Positive test: After examining nine low power fields, if 3 or more clumps of agglutinated red blood cells are observed (ie on average, equal to or greater than one clump per three low power fields) the test is positive and indicates the presence of a D positive fetal red blood cells in possibly significant numbers in the maternal blood.
2. Negative test: After examining nine low power fields if two or fewer clumps of agglutinated red blood cells are observed (ie on average, less than 1 clump per three low power fields) the test is negative, indicating that a large feto-maternal hemorrhage did not occur.

Note

1. The number of clumps observed may be influenced by several variables and should not be used as a means to determine the amount of feto-maternal hemorrhage.
2. The presence of rosettes or agglutination in the negative control tube indicates inadequate washing after incubation, allowing residual anti-D to agglutinate the D positive indicator cells.
3. For correct interpretation of the test results the test must be performed on the blood of a known D antigen negative mother of a recently delivered D positive child.
4. In cases of ABO incompatibility between the mother and child the mother’s natural ABO antibodies may destroy any fetal cells in the maternal blood specimen before testing is performed.
5. Failure to carry out the washing stages properly may give rise to a false positive result.