Managing a patient with whose post partum blood specimen tests positive in a rosette screening test for fetal-maternal hemorrhage, but tests negative in a Kleihauer-Betke (KB) or a flow cytometry test for the presence of Hgb F containing red cells

A colleague in Maine reports that her hospital laboratory has encountered instances where screening for fetal-maternal bleeding has been positive using a 'rosette test' (when testing for the presence of Rh positive red cells in a sample of Rh negative maternal blood), but a follow up Kleihauer-Betke (KB) test or a flow cytometry test has been negative for the presence of Hgb F containing red cells. She is aware that from time to time there can be a discordance between a rosette test result and follow up testing if a newborn's red cells have a preponderance of adult hemoglobin at birth. Most recently they had a case where an Rh-negative mother just delivered an Rh-positive newborn. The mother had no history of recent transfusion, transplantation or stem cell therapy, and there was no reason to believe she was a chimera. She had a negative direct antiglobulin test. A post-partum sample was positive in a rosette test and when that same sample was typed for Rh by an indirect antiglobulin test method, it tested weakly positive. Her pre-delivery blood sample was checked for D type by a direct agglutination method only. Flow cytometry done on the same post partum sample as was used for the rosette test was negative for Hgb F containing red cells. All QC for the rosette test, antiglobulin tests, Rh typing and flow cytometry showed expected performance. The QC for flow cytometry was performed at three levels using Fetal trol by Trillium Diagnostics. Ranges were: L1 [0.0 - 03%]; L2 [0.12-0.18%]; L3 [1.12-1.68%]. All results were within range on the day of patient tested. They have not done a standardized dilution curve using fetal red cells diluted in adult red cells because of the low value tested with the L1 control. Their CAP proficiency testing performance for flow cytometry has been acceptable. On the last survey the lower challenge had a mean of 0.47%. The lab recovered 0.4%. There have been no consistent biases one way or the other over the last three surveys. All results have been well within +/-2.0 SDI (usually less than 1.0 SDI). Also, patient samples tested by flow cytometry are stored at refrigerated temperature and typically run within 24 hours. She wonders if others have had similar cases, and if so, how were they resolved?

The following comments have been received.

ADDENDA October 15, 2007

1. A transfusion medicine physician in North Carolina points out that on page 794 of the15th edition of the AABB Technical Manual it states that "a strongly positive result is seen with red cells from a woman whose Rh phenotype is weak D rather than D-" Based on the information provided by the inquiring colleagues, his guess, in this case, is that the mother might have a weak D phenotype. Alternatively, the presence of rosettes or agglutination in the rosette test.... can indicate inadequate washing after the incubation step of the rosette test, allowing residual anti-D to agglutinate the D+ indicator cells. Thus, the possibility of a false positive rosette test must be ruled out.
   
   
2. The scientific director of a laboratory specializing in molecular blood group and platelet antigen testing reports that one possible explanation for the findings in this case is that the mother has a weak D phenotype. She acknowledges that the colleagues who initiated this discussion indicate that the strength of this patient’s rosette test was not particularly strong, and that on page 794 of the 15th edition of the AABB Technical Manual it states "a strongly positive result is seen with red cells from a woman whose Rh phenotype is weak D rather than D”. However, she points out that the Del phenotype requires adsorption-elution to be detected serologically, and even some weak D type 2 that are weak positive in the indirect antiglobulin test (IAT) when the C-antigen is in a Cis configuration could potentially react in a manner similar to this particular case report. Having said the above, she adds that a positive 'rosette test' followed by a negative Kleihauer-Betke (KB) (or flow cytometry) test can result if the mother has a weak D antigen not readily detected by IAT (weak D type 2, 5, 11, or 17 in her experience). Distinguishing maternal RBC weak D expression from the presence of fetal Rh+ cells in the maternal sample is one of the reasons confirmatory KB (or flow cytometry) testing is done, and this possibility should be considered when there is no evidence for a large fetal-maternal bleed. She concludes advising that molecular testing would be useful to confirm the D status of the mother.
   
   
3. An experienced immunohematologist who is affiliated with a University Medical Center in Michigan comments that one possible explanation for the findings in this case is that the mother has a weak D phenotype. The incidence of such phenotypes may be as high as 0.4%. He acknowledges that the colleagues who initiated this discussion indicate that the strength of the rosette test was not particularly strong, and that the red cells of some weak D positive mothers react very strongly positive in the rosette test. However, given the variability of D expression within weak D and partial D phenotype individuals, he has not seen that many cases of positive rosette tests in weak D and partial D individuals to be able to use the strength of the rosette test reaction to influence his opinion.

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