April 2023 – Cellular Therapy and Transfusion Medicine

Mayo Clinic Laboratories sends a blood sample to the lab with an order to perform a Fetomaternal Bleed, Flow Cytometry test. The mother is Rh negative, and the team needs to know how much RhIg to administer. When the sample is run, the histogram shows a distinct cell peak (B) of 20.23% as shown in Figure 1.

Figure 1
Figure 2

In Figure 1, which cell population is represented by peak B and how much RhIg is needed?

  1. Peak B denotes Adult F cells. Mother likely has HPFH-hereditary persistence of fetal hemoglobin. No additional testing is needed. Administer 1 vial of RhIg.
  2. Peak B denotes Adult F cells. Order Dual Antibody Fetal Cell Count testing.
  3. Peak B denotes fetal cells. Perform a Kleihauer Betke test to quantify fetal cells.
  4. Peak B denotes the fetal cells. Contact the team and inform them that a large fetomaternal bleed has occurred and provide RhIg dose required.

The correct answer is ...

Peak B denotes Adult F cells. Mother likely has HPFH-hereditary persistence of fetal hemoglobin. No additional testing is needed. Administer 1 vial of RhIg.

Figure 1

(A) % Adult cells = 79.71

(B) % Adult F cells = 20.23

(C) % Fetal cells = 0.06

Adult cell: Peak A: RBC population that does not contain Fetal Hemoglobin.

Adult F cells: Peak B: Dim-fluorescing cells that do not stain as brightly as true fetal cells: these are RBCs that contain a small amount of HbF but are not true fetal cells. This “shoulder” will be elevated in hereditary persistence of fetal hemoglobin.

Fetal cells: Peak C. True fetal cell population containing Hemoglobin F and stains brightly positive.

Fetal hemoglobin (hemoglobin F, HbF, alpha2gamma2) is the most abundant hemoglobin during gestation and accounts for 60 to 80 percent of total hemoglobin in the full-term newborn. It is almost completely replaced by adult hemoglobin (hemoglobin A, HbA, alpha2beta2) by 6 to 12 months of age in individuals without hemoglobinopathies. Eventually, in adulthood, it accounts for less than 1% of total hemoglobin. Hereditary persistence of fetal hemoglobin (HPFH) is a benign condition in which significant fetal hemoglobin production continues into adulthood, bypassing the normal shutoff point after which only adult-type hemoglobin should be produced.

In this case the Adult F cells was easily distinguished from true fetal cell gate, which contained 0.06% (C) and further testing was not necessary.

The dosing of RhIg is based on the number of fetal cells in the maternal circulation. The rules, however, provide a safety margin of 1 additional vial based on rounding so even if the calculation of RhIg suggests 0, 1 vial of RhIg is always given if the mother is RhNeg and the child is RhPos. For example:

Calculating the RhIG Dose: % Fetal Bleed x 5000 ml  +1 

                              0.0006 X 5000/30= 0.1(round down) + 1  = 1.0 vial of Rhig

Incorrect answers explanation:

Large amounts of Adult F cells are seen in hereditary persistence of fetal hemoglobin (HPFH) in which the shoulder of HPFH cells (Adult F cells) encroach on the true fetal cell gate. Refer to Figure 2(a) where peak for gate B (Adult F cells 26.21%) extends into gate C (True fetal cells 4.50%). This can interfere with an accurate determination of the fetal cells. The consequences of missing a fetal bleed are significant and can result in an immunizing event leading to hemolytic disease of the newborn in subsequent pregnancies. Therefore, it is imperative to count these cells accurately and dose the appropriate amount of RhIg.

In a mother with known HPFH, this can make it difficult to determine a FMB accurately. Most labs use a single antibody (anti-fetal hemoglobin, HbF) kit to detect fetal hemoglobin. With only one single antibody, there are limitations with the samples that have significant adult F cells containing HbF overlapping with the true fetal cells.

A Dual Antibody Fetal Cell Count Kit includes antibodies for hemoglobin F (HbF) and carbonic anhydrase (CA) providing additional discrimination between the fetal and maternal HbF+ cells in mothers with underlying HPHF. The kit uses anti-carbonic anhydrase (CA) and anti-fetal hemoglobin (HbF). Fetal RBCs will be negative for CA (HbF+CA-) while the mother’s RBCs with high amount of fetal Hb will stain positive for both HbF and CA. (HbF+CA+).

See Figure 2

Anti-HbF is used on the Y axis and Anti-CA on the X. Thus, relevant true fetal bleeds Fetal cells would be in the LUQ (HbF+, HbCA-). In this case they represent 0.25%.

In the lower right a second antibody to carbonic anhydrase (Anti CA) is used to distinguish Adult cells from fetal cells (HBF-, CA+) (78.74%).

Adult F cells with increased amounts of fetal Hb seen in HPFH are in the RUQ (Right upper quadrant) (HbF+, CA+) (20.38%).

Adding a secondary antibody such as carbonic anhydrase (CA) improved the delineation of fetal cells from 4.50% in Fig 2a to 0.25% fetal cells shown in the LUQ in Fig 2b. This demonstrates the utility of CA in discriminating fetal and maternal HbF+ cells in mothers with an underlying hereditary persistence of fetal hemoglobin.

Peak C, not Peak B denotes fetal cells and although the Kleihauer-Betke assay is a quantification test, it is not able to reliably separate maternal and fetal HbF+ cells and would not be useful in a maternal sample with an underlying hemoglobinopathy.


  1. Sharma DC, Singhal S, Woike P, Rai S, Yadav M, Gaur R. Hereditary persistence of fetal hemoglobin. Asian J Transfus Sci. 2020 Jul-Dec;14(2):185-186. doi:10.4103/ajts.AJTS_71_16. Epub 2020 Dec 19. PMID: 33767547; PMCID: PMC7983139. https://pubmed.ncbi.nlm.nih.gov/33767547/.
  2. Clark G, Svensson AM, Lieberman L. Perinatal issues in transfusion practice. Technical Manual. 20th Ed. Bethesda (MD); 2020:659-672.
  3. Krywko DM, Yarrarapu SNS, Shunkwiler SM. Kleihauer Betke Test. 2022 Aug 8. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 Jan–. PMID: 28613626. https://pubmed.ncbi.nlm.nih.gov/28613626/.

Saadiya Nazli, M.B.B.S.

Fellow, Cellular Therapy
Mayo Clinic

Margaret (Maggie) DiGuardo, M.D.

Consultant, Transfusion Medicine
Mayo Clinic
Assistant Professor of Laboratory Medicine and Pathology
Mayo Clinic College of Medicine and Science

MCL Education

This post was developed by our Education and Technical Publications Team.