Detection of Fetal Red Cells in Fetomaternal Hemorrhage Using a Fetal Hemoglobin Monoclonal Antibody by Flow Cytometry

Abstract

BACKGROUND:The laboratory determination of the level of fetal cells in maternal circulation remains an important support in the obstetrical management of women with suspected uterine trauma and in the proper dose administration of anti-D for prevention of Rh hemolytic disease of the newborn. Limitations in the sensitivity and precision of the widely used manual Kleihauer-Betke test have prompted an increased utilization of flow cytometric methods for fetal cell detection in maternal blood samples. STUDY DESIGN AND METHODS: Murine monoclonal antibodies directed against fetal hemoglobin (HbF) were developed, conjugated to fluorescein isothiocyanate, and used in a multiparametric flow cytometric assay developed for the quantitation of fetal red cells. A rapid intracellular staining method using brief glutaraldehyde fixation and Triton X-100 permeabilization prior to monoclonal antibody incubation was developed, along with optimization of the flow cytometric analysis protocol for the analysis of 50,000 cells. The performance of the assay was assessed for linearity and precision and correlated with the Kleihauer-Betke acid elution method. RESULTS: The anti-HbF flow cytometric method showed good correlation with the Kleihauer-Betke method (P = 0.86) and superior precision with a CV c15 percent for blood samples with >0.1 percent fetal cells.Analysis of 150 blood samples from nonpregnant adults, including individuals with elevated HbF due to hemoglobinopathies and hereditary persistence of HbF, gave a mean value of 0.02 percent fetal cells, and all results were less than 0.1 percent. CONCLUSIONS The anti-HbF flow cytometric method for detection of fetal cells offers a simple, reliable, and more precise alternative to the Kleihauer-Betke manual technique for the assessment of fetomaternal hemorrhage. The method has additional potential applications for the study of HbF levels or frequency of adult red cells with low levels of HbF (F cells) in individuals with hemoglobinopathies. he detection of circulating fetal cells in maternal blood represents an important area of laboratory support to the obstetrical management of ABBREVIATIONS: ELISA = enzyme-linked immunosorbent assay; FMH = fetomaternal hemorrhage; HAT = hypoxanthine aminopterin thymidine; Hb = hemoglobin; HbA = hemoglobin A; HbF = hemoglobin F; PBS = phosphate-buffered saline; PBS-BSA = PBS with bovine serum albumin; RBC(s) = red cell(s); RhIG = Rh immune globulin; WBC(s) =white cell(s). From the Department of Clinical Pathology, William Beaumont Hospital, Royal Oak, Michigan.

Automated quantitation of fetomaternal hemorrhage by flow cytometry for HbF‐containing fetal red blood cells using probability state modeling

“…These 'low' values artificially inflate the imprecision between the WinList replicate analyses. If these values are excluded, then the CVs would be approximately 4% and 7% for the manual intra-analyst and interanalyst analyses, respectively, which is in keeping with previous reports [6,[9][10][11]. If such variation is observed in expert analyses, we anticipate that it would even higher for nonexperts, where CV is typically reported in the range of 10-30% from regional proficiency testing programs for FMH testing by flow cytometry, such as those run by the College of American Pathologist or UK NEQAS programs.…”

Reference Values of Fetal Erythrocytes in Maternal Blood During Pregnancy Established Using Flow Cytometry

“…21 Flow cytometric methods are equally sensitive and have much better precision, irrespective of whether they demonstrate fetal cells by their surface Rh(D) antigen or intracellular HbF content. [5][6][7] Moreover, anti-HbF-based flow cytometric methods enable objective discrimination between fetal RBCs and adult F cells, which generally is difficult in the K-B test. 9,11 This distinction is important because maternal F cells have been reported to increase during the second trimester of pregnancy, 22,23 and misidentification of F cells as fetal RBCs may easily occur in pregnant women with sickle cell disease, thalassemia, hereditary persistence of fetal hemoglobin, or a hematologic malignancy.…”

“…[2][3][4] Methods using monoclonal antibodies to hemoglobin F (HbF) were developed later. 5,6 All flow cytometric methods have better precision than the K-B technique, and, moreover, the HbF-based methods allow objective discrimination between fetal RBCs and adult RBCs with an increased content of HbF, so-called F cells. 5,7,8 In the K-B method, F cells are often difficult to distinguish from fetal RBCs.…”

“…5,6 All flow cytometric methods have better precision than the K-B technique, and, moreover, the HbF-based methods allow objective discrimination between fetal RBCs and adult RBCs with an increased content of HbF, so-called F cells. 5,7,8 In the K-B method, F cells are often difficult to distinguish from fetal RBCs. Therefore, HbF-based flow cytometric methods can prevent false-positive results in patients with increased F-cell counts due to various hematologic diseases.…”

Diagnostic Dilemma of HbA1c Detection in Presence of a Hemoglobinopathy: A Case Report

“…A flow cytometric analysis based on the original procedure described by Davis et al [5] was enumerated on a ABBOTT Cell-Dyn Sapphire (Abbott GmbH & Co.KG, Wiesbaden, Germany) 1-colour flow cytometer, as a replacement for the Kleihauer-Betke test [6]. In brief, RBCs were fixed with glutaraldehyde and permeabilized by Triton X-100 (Sigma, St.Louis, MO), and a mouse monoclonal antibody against hemoglobin F was conjugated to Fluorescein isothiocyanate (FITC) from Caltag Laboratories, Burlingame, CA.…”