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

Principle

  • 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)

Controls

  • Negative control (normal adult FBC)

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

Screening

  • 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

Quantification

  • 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

Method

  • 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.

Controls

  • 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

Advisory

  • 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