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.

 

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

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)

-       Controls

o   Negative control (normal adult FBC)

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

-       Screening

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

-       Quantification

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

o   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

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.

-       Controls

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

-       Advisory

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