Severe fetomaternal hemorrhage confirmed and quantified by flow cytometry using anti fetal hemoglobin antibodies

Abstract

Severe fetomaternal hemorrhage (FMH) is a rare but potentially life-threatening perinatal complication (1). The traditional method for diagnosis of this situation is the acid elution (Kleihaur–Betke) test. However, this technique is relatively insensitive and non-specific, and does not allow quantification of the hemorrhagic process. We report a case of severe FMH confirmed and quantified by means of flow cytometric analysis using specific staining for cells containing fetal hemoglobin (HbF) in maternal circulation. We believe that this method is more sensitive, more specific and allows accurate quantification of FMH. A 30-year-old nullipara at 36 + 2 gestational weeks was admitted to the delivery room for decreased fetal movements. On admission, a sinusoidal fetal heart rate pattern was recorded (Fig. 1), and an immediate cesarean section was performed. A 2400 g female infant was delivered with Apgar scores of five and seven at 1 and 5 min, respectively. The infant was extremely pale, limp and bradycardic. Initial hematocrit and hemoglobin levels were 12.3% and 4.4 g/dL, respectively, while a direct Coombs' test was negative. She was treated initially by infusion of normal saline followed by packed red blood cells (20 cc/kg), until marked clinical improvement, and her hematocrit reached 35%. The rest of her postnatal course was unremarkable. Fetal cardiotogoram showing a sinusoidal fetal heart rate pattern. In an attempt to confirm the suspected diagnosis of FMH, we performed flow cytometric analysis of fetal erythrocytes in maternal blood samples obtained soon after delivery. The cells were washed, fixed permiabilized and stained with monoclonal antibodies against HbF according to the manufacturer's instructions (Bioatlantic, Nantes, France). Cell analysis was performed with a FACstarplus flow cytometer (Beckton-Dickenson, Mountain View, CA, USA). A 488-nm argon laser beam at 250 mV served for excitation. The threshold was set on forward light scatter to exclude platelets. Green fluorescence was measured using logarithmic amplification through a 530 ± 30 nm band-pass-filter. As a positive control we used 3 neonatal cord blood samples and as negative controls blood samples from five other women after normal deliveries. The positivity threshold was adjusted with positive controls. Almost 11% of maternal erythrocytes contained high HbF level, i.e. were of fetal origin (Fig. 2). These results were confirmed by analyzing the hemoglobin types in the hemolysate of the maternal blood using high performance liquid chromatography (results not shown). Histogram of flow cytometric analysis of three samples stained for HbF. The flourescence intensity is demonstrated on a log scale along the X-axis, and the cell count along the Y-axis. A. The presented case showing a major peak of HbF negative cells (maternal cells) and a minor peak of cells containing high levels of HbF, i.e, fetal cells. B. A sample of umbilical cord blood. C. A sample of control maternal blood. Detection of fetal cells in maternal circulation represents an important support for the clinical diagnosis of FMH. Most laboratories perform FMH estimates on the basis of the slide-based microscopic counting method of acid elution (Kleihaur–Betke) test. This technique is laborious and suffers from lack of sensitivity, subjectivity and precision (2). These limitations prompted the use of flow cytometry which overcomes all these limitations by rapid and sensitive cell-by-cell analysis (3). In the past, flow cytometry approaches of fetal cells in maternal blood relied mainly on detection of the human D antigen. However, this approach was applicable only to clinical situations with D antigen incompatibility and could not be utilized in all cases of FMH. Using antibodies to HbF as described in the current case allows for broad application to various clinical situations (4, 5). Moreover, the quantitative nature of flow cytometric analysis permits the distinction of true fetal cells, which contain HbF as the major form of hemoglobin, from maternal circulating F-cells that have lower cellular HbF content. This is particularly applicable for individuals with hereditary persistence of HbF or various hemoglobinopathies. To our knowledge, this is the first reported case of severe FMH confirmed and quantified by flow cytometry using anti HbF antibodies. Clinicians may wish to apply this rapid and accurate method for diagnostic purposes postnataly and even antenatally when clinical suspicion arises.