Estimation of FetoMateranl haemorrHage (FMH) (bsh 2009, BSH 2026)


Who to test

 

FMH estimation must be performed on:

  • Rh D- women, at least 30 minutes 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 sensitising events in Rh D- women after 20 weeks gestation

  • Specific scenarios when FMH testing should be performed:

    • Intrauterine fetal death / still birth, Trauma, Unexplained neonatal anaemia, Post intrauterine transfusion, Fetal blood sampling.

 

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 (and living donor bone grafts) when D+ donor into D- recipient

 

FMH testing is not required for:

  • Sensitising events prior to 20 weeks gestation (fetal blood vol., at this time is only 30ml)

  • 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 sensitising 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

Kleihauer (FMH) labelled.jpg

 

Acid Elution (modified Kleihauer-Betke Test)

Based on HbF vs HbA

Best for screening and 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

  • Scan the slide at low power using a x10 eyepiece and x10 objective

  • Examine 25 low power fields and count the fetal cells

  • 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

  • If result is >2ml —> flow cytometry

Limitations

  • Raised maternal HbF levels (2nd trimester, thalassaemia, sickle cell, HPFH) can cause large variation in density of staining —> intermediate cells which are hard to distinguish from fetal cells. Give a standard dose of anti-D and send for flow cytometry.

 

Flow Cytometry

Fluorochromes conjugated with IgG monoclonal anti-D

Most commonly used as quantification after positive acid elution finds >2ml FMH

Flow can also be used as a screening test in place of acid elution, not yet widely adopted

Method

  • Use same EDTA sample that was used for the screening test

  • Mix sample thoroughly

  • Wash cells to remove leukocytes and platelets

  • Use two markers (to exclude auto-fluorescent neutrophils)

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

Controls

  • Should include a mix of D+ and D- cells

  • Should discriminate between clinically significant bleed volumes (eg 0.2% vs 1% vs 10%)

  • Inert control should also 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

 

Discrepant results

Acid elution tends to overestimate fetal cells compared to flow cytometry.

Causes of discrepancies between the two methods:

  • D variants, HPFH, Sample storage or preparation, Assay technique, Limitations of testing methodology.

Acting on a discrepancy:

  • If acid elution and flow do not agree (after repeat testing) —> D type the fetus/neonate with cffDNA RHD genotyping or serological RBC D typing.

  • SOP for managing Anti-D prophylaxis is discrepancies occur

  • Incident reporting

Reporting results to clinicians

 

Reports need to be:

  • Timely – to ensure anti-D administered within 72 hours of the event

  • Clear

  • Advisory

Reports must therefore include:

  • Test method

  • The final result in ‘mL packed fetal RBCs’ rounded up to nearest 1ml

  • The recommended dose of Anti-D

  • Request for follow-up samples if required, including the time interval

Where possible, consider also including:

  • Reason for sample testing

  • Whether sample has been referred on for flow

  • Further details of effective administration of anti-D

  • Details of the person providing the clinical advice

  • Maternal D type

  • Information to explain discrepancies

  • Confirmation that no further samples are required

  • A statement on the tests Uncertainty of Measurement (UoM)