Estimation of FMH (2009)
Who to test
FMH estimation should be performed on:
- Rh D- women, following delivery of an Rh D+ baby
o 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:
- 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+
o Should be typed with routine saline reacting, IgM reagents that do not detect DVI.
o 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.
- Mother – EDTA for FMH, separate G&S tube (FMH underestimated after centrifugation of G&S samples)
- Baby – cord blood for ABO and D group
- 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
o HbF is more resistant to alkali denaturation and acid elution than HbA
o 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
o Thin, freshly prepared films easier to read (a 1:2 dilution may help)
o Modified test – elute only half the slide (allows comparison)
o Negative control (normal adult FBC)
o Positive control (cord blood added to adult whole blood in a 1:100 dilution
o 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)
o 25 low power fields should be screened
o If 10 or more fetal cells seen then quantification must be performed
o Less than 10 cells can be considered <2ml FMH
o Counted with aid of a Miller Square, counting minimum of 10,000 cells
o Mollinson formula then calculates the ml of FMH
- Fluorochrome conjugated with IgG monoclonal anti-D
- Best as a reference test after positive acid elution finds >2ml FMH
o Mix sample thoroughly
o Wash cells to remove leukocytes and platelets
o Two samples should be tested in tandem; discrepancy suggests error in sample preparation or flow counting.
o 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
o Whether supplementary anti-D is required if a standard dose has already been administered.
o The Anti-D dose required to cover the reported bleed
o Advice on follow-up testing to ensure clearance of D+ cells