Quantification Of Fetomaternal Hemorrhage Research Articles

Evaluation of Flow Cytometry and Kleihauer Techniques for Quantification of Fetomaternal Hemorrhage: A Prospective Cohort Study in Southwestern Iran

Background: Quantification of fetal red blood cells (RBCs) in maternal blood is of great importance to calculate appropriate dose of post-deliver anti D immunoglobulin in a rhesus D (RhD)-negative woman. Objective: The aim of this study is to evaluate a direct immunofluorescence flow cytometry technique in artificial and clinical samples and compared it to the Kleihauer-Betke test (KBT). Methods: This study was a prospective cohort design. Blood samples from 26 pregnant women who gave birth to RhD positive babies were tested using direct immunofluorescence flow cytometry and KBT techniques to determine the amount of FMH in the maternal circulation. The zone of D-positive cells was identified employing artificial samples including 0.3%, 0.6%, 1%, 1.5%, 2%, 5%, 10%, and 50% of D-positive fetal cells in D-negative maternal cells. Results: Analysis of 26 clinical samples for FMH showed consistent quantification with the flow cytometry and Kleihauer techniques. Although a good correlation was found between the KBT and flow cytometry results, in artificial samples containing more than 2% of fetal RhD positive cells, the flow cytometry results were closer to theoretical percentages. In a patient with FMH >4 mL, the FMH and consequently the required vial of Ig were overestimated using KBT. Conclusion: Most of the FMH calculated could have been neutralized by doses less than 625 IU, whereas the routine dose in Iran is more than double that amount (1500 IU). This achievement demonstrates that adjusting between the RhD immune globulin (RhDIg) dose and FMH size is inevitable.

The presence of F cells with a fetal phenotype in adults with hemoglobinopathies limits the utility of flow cytometry for quantitation of fetomaternal hemorrhage.

Detection and quantitation of fetomaternal hemorrhage (FMH) can be difficult in patients with pre-existing elevations of HbF, such as those with hemoglobinopathies. The aim of this study was to evaluate the utility of dual-color flow cytometry with the Fetal Cell Count Kit (FCCK) in differentiating adult and fetal HbF in this population, as compared to flow cytometry (FC) using HbF alone. Peripheral blood was obtained from normal adults and patients with hemoglobinopathies (β-thalassemia and sickle cell disease), including a small number of pregnant females. Cord blood was used to spike some samples with 5% fetal cells. Analysis by single color (HbF) and dual-color (HbF and carbonic anhydrase) FC was performed on these samples. Fetal cells were defined as those with high HbF fluorescence on single-color FC, and those that were HbF + CA- using the FCCK. The quantity of fetal cells detected by each technique was compared. Forty-six adult patients were included. In non-pregnant adults with hemoglobinopathies, a population of red cells with a fetal cell phenotype were detected by both techniques. The dual-color method reported lower quantities of these cells. In nineteen samples spiked with cord blood the FCCK consistently underestimated the quantity of fetal cells. Patients with β-thalassemia and sickle cell disease have a population of HbF-containing cells which are phenotypically similar to fetal cells. Even with dual-color flow cytometry (FCCK), the detection and quantification of FMH by flow cytometry in this population remains difficult. © 2017 International Clinical Cytometry Society.

Evaluation of FMH QuikQuant for the detection and quantification of fetomaternal hemorrhage

The Kleihauer-Betke test (KBT) is the most widely used assay for fetomaternal hemorrhage (FMH) detection in rhesus D negative women. In the current study, we sought to evaluate the performance of a flow cytometry (FCM) kit (FMH QuikQuant) using an anti-HbF antibody. Eighty-three pregnant women, 58 umbilical cord blood (UCB) dilutions in adult blood, and 6 control samples were tested in parallel with FCM and KBT. Firstly, we compared for each assay, on the 58 UCB preparations, results obtained to dilutions prepared. FCM results showed an excellent correlation (r = 0.97), a high reproducibility with a coefficient of variation lower than 20% for values reaching 10 fetal RBCs. KBT values were correlated (r = 0.94) but exhibited a poor reproducibility. Then, we compared both techniques on all samples. FCM showed a good correlation with KBT (r = 0.87) but the KBT exhibited a systematic overestimation of the FMH. For 8 out of 83 pregnant women, KBT was positive. Five were concordant with FCM results (KBT+/FCM+). On the three discordant (KBT+/FCM-), 2 were finally classified as false positive of the KBT because a second control sample was negative and additionally, FCM identified an increased rate of F cells. One discordant case (KBT+/FCM-) remained unexplained. By receiver operating characteristic analysis, we found a threshold at 4.5 RBCs for FCM with a sensitivity of 89.8% and a specificity of 93.2%. The FMH QuikQuant kit is a reliable and highly reproductive FCM method for FMH quantification.

Detection of fetomaternal hemorrhage

The prevention of Rhesus D alloimmunization through Rh immune globulin (RhIg) administration is the major indication for the accurate detection and quantification of fetomaternal hemorrhage (FMH). In the setting of D incompatibility, D-positive fetal cells can sensitize the D-negative mother, resulting in maternal anti-D alloantibody production. These anti-D alloantibodies may lead to undesirable sequelae such as hemolytic disease of the newborn (HDN). Since the widespread adoption of FMH screening and RhIg immunoprophylaxis, the overall risk of Rh alloimmunization and infant mortality from HDN has substantially decreased. The rosette screen, the initial test of choice, is highly sensitive in qualitatively detecting 10 mL of fetal whole blood in the maternal circulation. As the screen is reliant on the presence of the D antigen to distinguish fetal from maternal cells, it cannot be used to detect FMH in D-positive mothers or in D-negative mothers carrying a D-negative fetus. The Kleihauer-Betke acid-elution test, the most widely used confirmatory test for quantifying FMH, relies on the principle that fetal RBCs contain mostly fetal hemoglobin (HbF), which is resistant to acid-elution whereas adult hemoglobin is acid-sensitive. Although the Kleihauer-Betke test is inexpensive and requires no special equipment, it lacks standardization and precision, and may not be accurate in conditions with elevated F-cells. Anti-HbF flow cytometry is a promising alternative, although its use is limited by equipment and staffing costs. Hematology analyzers with flow cytometry capabilities may be adapted for fetal cell detection, thus giving clinical laboratories a potentially attractive automated alternative for quantifying FMH.

Quantification of Feto-Maternal Hemorrhage: Selection of Techniques for a Resource-Poor Setting

Background: Though RhD sensitization in RhD-negative mothers is now almost eradicated in the developed world, it continues to be a major health problem in developing nations like India. Inadequate immunoprophylaxis is the main reason. Adequate dose calculation of anti-D Ig is possible through estimation of correct feto-maternal hemorrhage (FMH) volume. In this regard, different methods have been used. Methods: We evaluated three quantitative techniques of estimating FMH: the Kleihauer-Betke test (KBT) and two flow cytometry (FC) techniques, i.e., the indirect immunofluorescence technique (IIFT) and direct immunofluorescence technique (DIFT). Stock solutions of both RhD-positive and D-negative cells were made, and 7 serial dilutions of RhD-positive cord cells in D-negative adult cells were prepared. Result: Both KBT and FC approximated the expected concentration of fetal RhD-positive cells in all mixtures tested. In both methods, an underestimation of fetal RhD-positive cells was observed when their expected concentration was ≧0.75%. Conclusion: Though FC is the most sensitive of all techniques, very few laboratories in developing nations can afford such a costly device, so it will be prudent for them to use KBT as the gold standard due to its rapidity, simplicity and affordability.

Inaccurate Doses of Rh Immune Globulin After Rh-Incompatible Fetomaternal Hemorrhage: Survey of Laboratory Practice

Abstract Context.—Rh(D)-negative women with a large fetomaternal hemorrhage (FMH) from an Rh(D)-positive fetus are at risk for anti-D alloimmunization if they do not receive adequate Rh immune globulin (RhIG). Determination of the adequate RhIG dose for these women is a critical laboratory procedure for protecting their future Rh(D)-positive children. Objective.—To determine how often laboratories recommended an inaccurate dose of RhIG for excess FMH. Design.—Nearly 1600 laboratories using the College of American Pathologists' proficiency testing for fetal red blood cell detection were surveyed to determine (1) their calculation method and (2) the number of RhIG doses recommended for a survey specimen, based on their measured percentage of fetal red blood cells. We surveyed nearly 1450 laboratories for their accuracy in determining RhIG dose, using 2 common calculation methods we provided. Results.—The AABB Technical Manual method was used by 67% of responding laboratories. However, 20.7% of laboratories using this method would have recommended an inaccurate dose of RhIG—11.5% too much and 9.2% too little—for the level of FMH reported in the survey specimen. If all laboratories had used the common recommendation of 300 μg/30 mL of fetal blood present, 2% would have recommended RhIG doses too low for the volume of FMH they measured. In 3 of the 4 calculation exercises we provided, 20% to 30% of laboratories underestimated the necessary dose of RhIG. Conclusions.—Based on our surveys, some mothers with excess FMH may be receiving inaccurate doses of RhIG. Laboratories performing quantification of FMH should review their procedures and training for calculating RhIG dosage.

Assessment of Fetomaternal Hemorrhage by Flow Cytometry and Kleihauer-Betke Test in Rh-Negative Pregnancies

Objectives: To assess the efficacy of flow cytometry (FC) in the detection and quantification of fetomaternal hemorrhage (FMH) in comparison to the Kleihauer-Betke test (KBT). Methods: 25 unsensitized Rh-negative mothers who had delivered Rh-positive infants were included. Presence of FMH was determined by KBT and FC using FITC-labeled BRAD-3 antibodies. Results: FMH was detected in 19 (76%) patients by FC and 23 (92%) patients by KBT prior to delivery, and in 21 (84%) patients by FC and 23 (92%) patients by KBT after delivery. The mean volume of FMH in the post-delivery samples by KBT and FC were 0.34 ± 0.26 ml (range 0.05–1.2 ml) and 0.37 ± 0.57 ml (range 0.02–2.6 ml) respectively. The volume of post-delivery FMH estimated by KBT and FC correlated well (r = 0.75; ICC α = 0.73). A higher agreement between KBT and FC was seen in the 0.1–0.5 ml range (ĸ = 0.65; p < 0.01). Conclusions: Both manual KBT and FC using FITC-BRAD-3 antibodies show good sensitivity in detecting and quantifying fetal red cells. There is a good correlation between the methods in the 0.1- to 0.5-ml range of FMH.

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

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.

The Quantification of Fetomaternal Hemorrhage by an Enzyme‐Linked Antibody Test with Glutaraldehyde Fixation

A modified enzyme-linked antiglobulin test (ELAT) for quantifying D-positive red blood cells in the circulation of D-negative pregnant women is described. The antibody-antigen bond was stabilized and the problem of hemolysis eliminated by the use of 0.05% glutaraldehyde. The r value for the standardization curves for measuring cord RBC in mixtures was 0.98; coefficient of variation 5.8%. Reproducible quantitation of 0.125% D-positive cord RBC mixed with D-negative RBC was demonstrated. In comparative studies of artificial mixtures, Kleihauer-Betke results for more than 1% cord RBC were 50-100% greater than the true values. In several examples of large fetomaternal hemorrhages, the ELAT results matched the clinical data more closely than the Kleihauer-Betke based estimates. 2 ml of fetal RBC in a 1,600-ml red cell mass can be quantified using the modified ELAT.

The Quantification of Fetomaternal Hemorrhage by an Enzyme-Linked Antibody Test with Glutaraldehyde Fixation

A modified enzyme-linked antiglobulin test (ELAT) for quantifying D-positive red blood cells in the circulation of D-negative pregnant women is described. The antibody-antigen bond was stabilized and the problem of hemolysis eliminated by the use of 0.05% glutaraldehyde. The r value for the standardization curves for measuring cord RBC in mixtures was 0.98; coefficient of variation 5.8%. Reproducible quantitation of 0.125% D-positive cord RBC mixed with D-negative RBC was demonstrated. In comparative studies of artificial mixtures, Kleihauer-Betke results for more than 1% cord RBC were 50-100% greater than the true values. In several examples of large fetomaternal hemorrhages, the ELAT results matched the clinical data more closely than the Kleihauer-Betke based estimates. 2 ml of fetal RBC in a 1,600-ml red cell mass can be quantified using the modified ELAT.