Estimation of FetoMateranl haemorrHage (FMH) (bsh 2009)

Who to test


FMH estimation should be performed on:

  • Rh D- women, following delivery of an Rh D+ baby

    • Babies should be typed with routine saline reacting, IgM reagents that do not detect DVI. Weak D or Variant D should be treated as D+ for purpose of anti-D administration.

  • Following all potentially sensitizing events in Rh D- women after 20 weeks gestation


Flow cytometry for minor D+ population may be required:

  • Following Rh D+ RBC transfusion to a Rh D- woman of childbearing potential to estimate or confirm the dose of anti-D required.

  • In solid organ transplant when D+ donor into D- recipient


FMH testing is not required for:

  • Non-severe sensitizing event prior to 20 weeks gestation

  • When woman is known to have immune anti-D

  • When fetus/baby known to be D-

  • When woman is D+

    • Should be typed with routine saline reacting, IgM reagents that do not detect DVI.

    • If testing D+ with these reagents then unlikely to make an anti-D that will adversely affect the baby

  • In D+ women with unexplained abdominal pain in late pregnancy, FMH by acid elution is of limited diagnostic use. Better tests are available for suspected placental abruption.


Samples Required


At delivery

Mother – EDTA for FMH, separate G&S tube (FMH underestimated after centrifugation of G&S samples)

Baby – cord blood for ABO and D group


During pregnancy

Maternal EDTA for FMH estimation following sensitizing events after 20 weeks gestation

Up to 28 weeks, maternal G&S should be performed prior to each anti-D dose

After 28 weeks, anti-D is still required even if RAADP given, but antibody screening is not.


Methods of FMH Estimation


Acid Elution (Kleihauer Test)

Based on HbF vs HbA

Best for initial quantification of FMH


  • HbF is more resistant to alkali denaturation and acid elution than HbA

  • Fixed, dry blood film placed in acid buffer, HbA is denatured and eluted leaving behind ghost cells. Cells containing HbF are stainable and stand out in a sea of maternal ghost cells.

Slide prep

  • Thin, freshly prepared films easier to read (a 1:2 dilution may help)

  • Modified test – elute only half the slide (allows comparison)


  • Negative control (normal adult FBC)

  • Positive control (cord blood added to adult whole blood in a 1:100 dilution


  • A low power field using a x10 eyepiece and x10 objective will show a minimum of 1600 red cells (figure extrapolated from 100 cells seen with a x10 eye, x40 obj)

  • 25 low power fields should be screened

  • If 10 or more fetal cells seen then quantification must be performed

  • Less than 10 cells can be considered <2ml FMH


  • Counted with aid of a Miller Square, counting minimum of 10,000 cells

  • Mollinson formula then calculates the ml of FMH


Flow Cytometry

Fluorochrome conjugated with IgG monoclonal anti-D

Best as a reference test after positive acid elution finds >2ml FMH


  • Mix sample thoroughly

  • Wash cells to remove leukocytes and platelets

  • Two samples should be tested in tandem; discrepancy suggests error in sample preparation or flow counting.


  • Inert control should be used to determine the proportion of background non-specific uptake of fluorochromes, which can be subtracted for the count of anti-D fluorochrome cells


Reporting results to clinicians


Reports need to be:

Timely – within 72 hours

Clear – report result in ‘mL of fetal red cells’ rounded up to nearest 1ml


  • Whether supplementary anti-D is required if a standard dose has already been administered.

  • The Anti-D dose required to cover the reported bleed

  • Advice on follow-up testing to ensure clearance of D+ cells