A paper was recently published in Transfusion Medicine by Kennedy GA et al. entitled "Quantification of feto-maternal haemorrhage (FMH) by flow cytometry anti-fetal haemoglobin labeling potentially underestimates massive FMH in comparison to labeling with anti-D" (Transfus Med 2003;1325-33).

Since a flow cytometric approach using anti-HbF is now available in the USA, one e-network member has inquired whether others in the forum have any comments on this report, or reservations about using this test.

The following responses have been received.

1. A colleague in Ohio wonders if laboratories using a flow cytometry method to quantititate FMH are calculating the dose of RHIG by rounding up the calculated number of vials and then adding one vial to the calculated dose, as is done by many laboratories when using a Kleihauer-Betke test. If not, what calculation is being used to determine the correct dose of RHIG to provide adequate immunoprophylaxis when using a flow cytometry method to determine the size of a feto-maternal bleed?

ADDENDA Jan 29, 2005

2. Dr. Bruce H. Davis of Trillium Diagnostics, LLC in Maine (attribution used with permission) cautions readers of the above referenced paper by Kennedy and colleagues that the authors reported using a low concentration of the anti-HbF antibody in their study, hence at higher levels of fetal cells the fetal RBC staining was undersaturated resulting in their reported findings. Hence the study's conclusion should not be considered a condemnation of the anti-HbF method, but rather the result of the flow cytometry technique that was reported by the authors. Dr. Davis suggests that laboratories should always verify that the concentration of antibody used in flow cytometry will result in no change in fluorescence intensity of fetal RBCs in the range of 0 - 15% fetal cells by testing mixtures of cord blood spiked into adult blood. In his hands, he reports that this is typically seen with 10 microliters of the Caltag anti-HbF product (PDF product insert). Other lines of evidence that the HbF method is valid are found in the CAP survey (HBF) where there has never been evidence that the anti-HbF method undercounts fetal cells compared to the Kleihauer method. Additionally, a stabilized control product is available for FMH assays called Fetaltrol (PDF product insert) that is composed of Rh positive fetal cells in Rh negative adult cells. Every lot of this product is reportedly tested by both anti-D and anti-HbF flow cytometry, in order to demonstrate no difference between the two methods in either the low positive control (0.15% fetal cells) or high positive control (1.5% fetal cells) levels. In summary, in Dr. Davis' opinion the anti-HbF method for FMH, which currently is the only FDA cleared method for fetal cell counting, has been validated in multiple labs over the last five years and found to perform in a reliable fashion superior to the Kleihauer assay, at least when performed properly.

ADDENDA July 9, 2007

3. A California colleague reports that his laboratory currently performs a Kleihauer Betke staining of fetal cells in order to determine the need of additional RhIG, or to examine whether there has been a serious trauma to a fetus following an impact injury to a pregnant woman's abdomen. The inquiring colleague wonders if any transfusion services perform the measurement of fetal hemoglobin in a maternal sample by using a chemistry analyzer? For example, his laboratory has a Tosoh machine that quantitates fetal hemoglobin in a sample. He wonders if this equipment could be used in place of the staining method to measure a fetal-maternal bleed?

ADDENDA July 11, 2007

4. A transfusion medicine physician in San Antonio, Texas reports having reviewed the CLSI (Clinical and Laboratory Standards Institute, formerly NCCLS) Approved Guideline on Fetal Red Cell Detection H52-A and did not see any mention of using a chemistry analyzer. Based on her extensive experience with fetal-maternal hemorrhage (FMH) detection, she feels that while the use of a chemistry analyzer to detect such bleeding might seem intuitively interesting, on reflection it is not appropriate. FMH testing is done to determine the volume of Rh positive fetal red cells that are circulating in an Rh negative mother to assure that the correct RhIg dose is offered, or to determine the volume of fetal blood in a maternal circulation to estimate the danger of exsanguination to a fetus. A very small proportion of fetal cells in a maternal circulation (less than 6%) is equivalent to the entire blood volume of a full term infant. In most circumstances one is looking at FMH that constitute less than 2% of the maternal circulation. The normal fetal hemoglobin present in an adult can range up to 2%. How can one differentiate the fetal hemoglobin present in the mother from the one that comes from fetal cells? She does not think it is possible. And then how would one correlate the % hemoglobin F with an actual volume of red cells!! Honestly she does not see how this could work.

Please submit comments to the e-Network Forum.

Ira A. Shulman, MD
CBBS e-Network Forum Editor & Moderator