HPLC and flow cytometry combined approach for HbF analysis in fetomaternal haemorrhage evaluation.

Introduction: Recently, a flow cytometric (FC) based test has been developed for detection of circulating fetal cells to replace the less accurate and reproducible Kleihauer-Betke test.FC test is easier to perform, it can distinguish the origin of fetal cells, but it is expensive and available in highly specialized laboratories. We evaluated the introduction of high-performance liquid chromatography (HPLC) approach as initial screening to identify patients who need an additional FC test to better discriminate the nature of haemoglobin-F (HbF) positive cells. Methods: Blood samples from 130 pregnant women suspected to have fetomaternal haemorrhage were analysed with HPLC and FC methods. The cut-off for HbF HPLC concentration was calculated. Statistical analyses for the evaluation of HPLC as a screening method were performed. The positivity cut-off of HbF to be used as decision-making value to continue the investigation was calculated. Results: An excellent agreement (R2 > 0.90) was observed between the percentage of HbF obtained by HPLC and the percentage of fetal cells detected by FC. Results obtained from each assay were compared to define the HPLC threshold below which it is not necessary to continue the investigations, confirming the maternal nature of the HbF positive cells detected. Our study demonstrated that a cut-off of 1.0 % HbF obtained by HPLC was associated with the lowest rate of false negative results in our patient cohort. Conclusions: This study provides a new FMH investigation approach that possibly leads to a reduction in times and costs of the analysis.

A Case of Massive Fetal-Maternal Hemorrhage: Lessons Learned in Diagnosis and Treatment.

The use of Rho(D) immune globulin in Rh-negative pregnant women has become standard of care, but many practicing clinicians do not know the dosing recommendations for this essential medication. In this article, we describe a case of a 15-year-old girl who presented with intrauterine fetal demise and was found to have massive fetomaternal hemorrhage. Kleihauer-Betke testing results indicated nearly 460 mL of fetal blood in the maternal circulation. The patient ultimately received 4800 µg of Rho(D) immune globulin, a dose that required close coordination with the obstetrical service and pharmacy. Although this is an unusual case of large-volume, potentially chronic, fetomaternal hemorrhage, it is also an excellent illustration of the principles for diagnosing this condition, as well as providing dosing guidelines for Rho(D) immunoglobulin to prevent alloimmunization.

Severe Neonatal Anemia Due to Spontaneous Massive Fetomaternal Hemorrhage at Term: An Illustrative Case with Suspected Antenatal Diagnosis and Brief Review of Current Knowledge.

Fetomaternal hemorrhage is defined as transfer of fetal blood into placental circulation and therefore into maternal circulation during pregnancy, and represents an important contributor to intrauterine fetal demise and neonatal death. The condition is rarely diagnosed prenatally because clinical findings are often nonspecific, and it is unpredictable. In this paper we present an illustrative case of massive spontaneous fetomaternal hemorrhage where the diagnosis was highly suspected antenatally based on maternal reported reduced fetal movements, abnormal suggestive cardiotocographic trace, and increased peak systolic velocity in the fetal middle cerebral artery. We discuss obstetrical and neonatal management and review the current knowledge in the literature. Maintaining a high index of suspicion for this condition allows the obstetrician to plan for adequate diagnostic tests, arrange intrauterine treatment or delivery, and prepare the neonatal team.

Evaluation of machine learning-driven automated Kleihauer-Betke counting: A method comparison study.

INTRODUCTION: The Kleihauer-Betke (KB) test is the diagnostic standard for the quantification of fetomaternal hemorrhage (FMH). Manual analysis of KB slides suffers from inter-observer and inter-laboratory variability and low efficiency. Flow cytometry provides accurate quantification of FMH with high efficiency but is not available in all hospitals or at all times. We have developed an automated KB counting system that uses machine learning to identify and distinguish fetal and maternal red blood cells (RBCs). In this study, we aimed to evaluate and compare the accuracy, precision, and efficiency of the automated KB counting system with manual KB counting and flow cytometry. METHODS: The ratio of fetal RBCs of the same blood sample was quantified by manual KB counting, automated KB counting, and flow cytometry, respectively. Forty patients were enrolled in this comparison study. RESULTS: Comparing the automated KB counting system with flow cytometry, the mean bias in measuring the ratio of fetal RBCs was 0.0048%, with limits of agreement ranging from -0.22% to 0.23%. Using flow cytometry results as a benchmark, results of automated KB counting were more accurate than those from manual counting, with a lower mean bias and narrower limits of agreement. The precision of automated KB counting was higher than that of manual KB counting (intraclass correlation coefficient 0.996 vs 0.79). The efficiency of automated KB counting was 200 times that of manual counting by the certified technologists. CONCLUSION: Automated KB counting provides accurate and precise FMH quantification results with high efficiency.

Systematic Kleihauer-Betke Test after External Cephalic Version for Breech Presentation: Is It Useful?

The incidence of fetomaternal hemorrhage (FMH) after external cephalic version (ECV) has been poorly reported. In this study, we evaluated the frequency of FMH, diagnosed by positive Kleihauer-Betke test (KBT), after ECV attempt and then evaluate the relevance of its routine use after procedure. A total of 282 women with a term breech presentation and who had ECV attempt were recruited from January 2014 and December 2018. After ECV attempt, women were systematically screened for FMH using KBT. Data on ECV attempt, KBT results, perinatal and neonatal outcomes were collected and compared between women with positive (cases) and negative KBT (controls) after ECV. The success rate of ECV was 22.0% (62/282). Eight women (2.9%) experienced transient fetal heart rate (FHR) abnormalities after ECV. In five (1.8%) women, KBT was positive after ECV. Obstetrical management was modified for two of these five women due to continuous positivity of KBT at day 1 and day 7 controls after ECV attempt. A cesarean section was planned 7 days earlier due to persistent high FMH on day 7 (6 mL fetal blood) in one woman and the labor was induced for persistent high FMH on day 7 (20 mL fetal blood) for another woman. No newborns have signs of fetal anemia at birth and there was no significant difference in neonatal status between two groups. FMH after ECV attempt are rare, and no negative fetal or neonatal outcomes were observed when KBT was positive, even strongly (>5 mL fetal blood). It appears that systematic KBT after attempted ECV is probably not useful.

Reduced fetal movement during pregnancy: Is the Kleihauer-Betke test really useful?

INTRODUCTION: Reduced fetal movement (rFM) is a frequent cause of consultation during the pregnancy and can reveal feto-maternal hemorrhage (FMH) that is sometimes responsible of severe fetal anemia. Our primary objective was to evaluate the contribution of the KBT in case of rFM. Our secondary objective was to compare it with ultrasound examination including peak systolic velocity of the middle cerebral artery (MCA-PSV) to predict neonatal anemia. MATERIALS AND METHODS: We conducted a retrospective study from January 2016 to December 2017 at Armand-Trousseau Hospital in Paris. We analyzed all patients consulting for rFM from 18 to 41 weeks of gestation. We compared the performance of KBT and MCA-PSV to predict neonatal anemia (Hemoglobin at birth under 13.5 g/dL) and severe neonatal anemia (Hb < 10 g/dL). RESULTS: Among the 338 patients, 327 KBT (96.7%) were performed. KBT was found positive in three cases (0.9%). Only one neonate (0.3%) presented with severe anemia requiring a postnatal transfusion. MCA-PSV was performed in 166 cases (49.1%). KBT and MCA-PSV were significantly correlated with neonatal hemoglobin at birth. KBT was better than MCA-PSV to predict neonatal anemia, while MCA-PSV was better than KBT to predict moderate to severe anemia. The KBT and MCA-PSV Doppler had excellent sensitivity and predictive negative values (100%), but they had poor predictive positive values for severe neonatal anemia. CONCLUSION: In case of decreased fetal movement, we suggest performing fetal cerebral Doppler. MCA-PSV could suffice in first approach. KBT may be performed if there is suspicion of fetal anemia in order to confirm FMH.

The prevalence of an elevated F cell population in a maternal and gynaecology cohort.

OBJECTIVES: This study aimed to determine F cell prevalence in a cohort of maternal and gynaecology specimens using QuikQuant anti-HbF flow cytometry (FC) kit and to investigate if the presence of maternal F cells can lead to fetomaternal haemorrhage (FMH) overestimation. BACKGROUND: The gold standard to estimate FMH is the Kleihauer-Betke test (KBT). The KBT has proved to be insufficiently sensitive to detect low numbers of circulating fetal cells due to the presence of maternal F cells. At present, the prevalence of false positive KBT results due to raised maternal F cell population, defined as >5%, is poorly characterised. METHODS: A total of 120 specimens were tested for the presence of F cells and fetal cells by KBT and anti-HbF FC. The results calculated were compared to determine FMH overestimation. RESULTS: Of our cohort, 32% showed an elevated F cell population, of which 69% (27 of 39) were clinically significant according to KBT (>2 mL FMH). The mean FMH volumes by KBT and anti-HbF FC were 3·90 mL (0·20-35·40 mL) and 4·09 mL (0·20-9·70 mL), respectively. CONCLUSION: The study highlighted that an elevated F cell level could be found in the cohort tested, with an F cell level of >10% causing significant FMH overestimation by KBT.

Anemia neonatal grave secundaria a transfusion feto-materna: a proposito de un caso / Severe neonatal anemia due to fetomaternal hemorrhage: an ilustrative case

La transfusión feto-materna es el paso de eritrocitos fetales a la circulación materna. Cuando es masiva, tiene una incidencia aproximada del 0,2-0,9 %. Generalmente, se desconoce el agente desencadenante, pero, en ocasiones, se pueden identificar factores de riesgo. En el embarazo, suele ser asintomática; el síntoma más frecuente es la disminución de los movimientos fetales (el 26 %) en relación con la anemia grave. Se diagnostica mediante la detección de hemoglobina fetal en la sangre materna (test de Kleihauer o citometría de flujo). Se presenta a un recién nacido con anemia crónica secundaria a la transfusión fetomaterna, que, después del tratamiento con transfusión de concentrado de hematíes, tuvo como complicación síntomas de sobrecarga de volumen y empeoramiento clínico. Tras la realización de una exanguinotransfusión, evolucionó favorablemente, sin secuelas.

Fetomaternal transfusion (FMT) is defined by the transfer of fetal blood into the maternal circulation. The incidence of massive FMT is estimated to be approximately 0.2-0.9 % of births. Although a number of etiologies have been associated with FMT, most causes remain unidentified and the pregnancy is usually asymptomatic. The most frequent symptom is the decrease in fetal movements (26 %) in relation to severe anemia. Several diagnostic modalities for FMT are described (Kleihauer stain, flow cytometry). We describe a case of a newborn with chronic anemia secondary to FMT who, after treatment with transfusions of red blood cells, presented volume overload and clinical worsening as a complication. In this case, our patient needed exchange transfusion for definitive improvement without disability.

[Severe neonatal anemia due to fetomaternal hemorrhage: an ilustrative case]. / Anemia neonatal grave secundaria a transfusion feto-materna: a proposito de un caso.

Fetomaternal transfusion (FMT) is defined by the transfer of fetal blood into the maternal circulation. The incidence of massive FMT is estimated to be approximately 0.2-0.9 % of births. Although a number of etiologies have been associated with FMT, most causes remain unidentified and the pregnancy is usually asymptomatic. The most frequent symptom is the decrease in fetal movements (26 %) in relation to severe anemia. Several diagnostic modalities for FMT are described (Kleihauer stain, flow cytometry). We describe a case of a newborn with chronic anemia secondary to FMT who, after treatment with transfusions of red blood cells, presented volume overload and clinical worsening as a complication. In this case, our patient needed exchange transfusion for definitive improvement without disability.

La transfusión feto-materna es el paso de eritrocitos fetales a la circulación materna. Cuando es masiva, tiene una incidencia aproximada del 0,2-0,9 %. Generalmente, se desconoce el agente desencadenante, pero, en ocasiones, se pueden identificar factores de riesgo. En el embarazo, suele ser asintomática; el síntoma más frecuente es la disminución de los movimientos fetales (el 26 %) en relación con la anemia grave. Se diagnostica mediante la detección de hemoglobina fetal en la sangre materna (test de Kleihauer o citometría de flujo). Se presenta a un recién nacido con anemia crónica secundaria a la transfusión fetomaterna, que, después del tratamiento con transfusión de concentrado de hematíes, tuvo como complicación síntomas de sobrecarga de volumen y empeoramiento clínico. Tras la realización de una exanguinotransfusión, evolucionó favorablemente, sin secuelas.

Usefulness of maternal fetal red blood cell count in rhesus-positive pregnant women.

Background Fetal red blood cells (FRBC) in maternal blood are counted in rhesus-negative women to determine the amount of anti-D immunoglobulin to be administered in the case of a rhesus-positive fetus. In rhesus-positive pregnant women this is done in not always very well-defined indications including trauma, miscarriage, fetal death and diminished fetal movements. The aim of this study is to determine if the detection of FRBC is useful in rhesus-positive pregnant woman. This was done by assessing maternal and fetal characteristics that are more likely to give a positive test. Materials and methods This was a retrospective cohort study. Results A total of 169 FRBC tests were performed in 161 rhesus-positive pregnant women. FRBC were found in 45 (26.6%) of the women. Three patients experienced a miscarriage although their FRBC tests were negative (p = 0.295). Of the seven patients who experienced unexpected stillbirths, three tested positive. The deaths were not less likely to occur if the results had been negative (p = 0.631). There was a statistically significant difference between the different types of trauma indications (p = 0.025): the test was more likely positive if there had been a fall on the ground or staircase or blunt trauma (p = 0.041, 0.026 and 0.018, respectively). FRBC were not more frequently present in the absence of fetal movements (n = 16, p = 0.693). Conclusion FRBC in maternal blood were more likely positive in the case of a fall on the ground, or from a staircase. However, a positive test does not necessarily imply fetal pathology and, therefore, does not contribute to clinical management.

[Trauma and pregnancy: Is the Kleihauer-Betke test really useful?] / Traumatisme et grossesse : le test de Kleihauer est-il vraiment utile ?

OBJECTIVE: To evaluate the pertinence of Kleihauer-Betke (KB) test, in case of abdominal trauma during pregnancy in forecast of fetal outcomes, according to trauma severity. METHODS: A single-center retrospective study conducted between January 2014 and April 2016 in a maternity type III and a trauma center, which included the pregnant women admitted for abdominal trauma. The trauma's severity was assessed using the guidelines of the Society of Obstetricians and Gynaecologists of Canada. The impact of a positive KB test, defined as>0.1%, was analyzed. Adverse outcome was defined as one or more of the following complications: intrauterine fetal death, placental abruption, pre-term birth<37 weeks of gestation, and fetal or neonatal anemia. RESULTS: During the study period, 265 pregnancies involved into an abdominal trauma were included: 69% with a minor trauma and 31% with a severe trauma. Of all patients, 5.6% presented a positive KB test, among then 15.4% had an adverse outcome. There was no significant difference in the rate of adverse outcomes in the positive KB group and the KB negative group either in the overall population (P=0.16), in the minor trauma population (P=1) or in the major trauma population (P=0.14). The predictive positive values were respectively in the global population, in the minor trauma group and in the severe trauma group 15.4%, 0% and 25%. CONCLUSIONS: The KB test does not seem to be useful in case of trauma during pregnancy to define adverse outcome.

Placental findings in feto-maternal hemorrhage in livebirth and stillbirth.

Feto-maternal hemorrhage (FMH) is not an uncommon event during pregnancy with important clinical implications for both maternal and fetal outcomes. The diagnosis is often made using Kleihauer-Betke (KB) test. As FMH occurs transplacentally, examination of the placenta may contribute to the diagnosis of FMH. This retrospective case-control study aims to examine the placental features associated FMH in patients with known positive KB test results. When compared with KB negative placentas (n=88), KB positive placentas (n=49) had significantly higher incidence of pallor (6/49 vs 0/88, p=0.0017), IVT (11/49 vs. 5/88, p=0.0032) and nRBCs (12/49 vs. 4/88, p=0.0008). Autopsy cases with fetal or neonatal death due to FMH, (n=13) compared to a cohort of 162 placentas associated with other, non-FMH causes of death also had significantly higher frequency of pallor (5/13 vs 0/162, p<0.0001), IVT (6/13 vs 24/162, p=0.011) and nRBCs (11/13 vs 67/162, p=0.003). Pallor and nRBC were also associated with higher volume of FMH. Placental parenchymal pallor, intervillous thrombi and presence of nRBCs are significantly associated with documented FMH in both normal pregnancies and pregnancies associated with fetal or neonatal death. The presence of these findings, especially in combination, may suggest the need for maternal KB testing to rule out FMH and neonatal monitoring and/or intervention.

Quantitative fetomaternal hemorrhage assessment with the use of five laboratory tests.

INTRODUCTION: In various countries, standard doses of anti-D IgG used for postpartum immunoprophylaxis of hemolytic disease of fetus and newborn (HDFN) vary from 100 µg to 300 µg. There are also different regulations concerning FMH assessment, and opinions about applicable tests are inconclusive. METHODS: Three flow cytometry tests (FCTs) with anti-D, anti-HbF, anti-HbF+CA antibodies, and two modifications of microscopic Kleihauer-Betke test (KBT) were used. RESULTS: In all artificial mixtures with known concentrations, FCTs and KBT with counting 10 000 RBCs had similar satisfying sensitivity and specificity. KBT with counting 2000 RBCs had to be disqualified because of significant discrepancies between expected and measured values of FMH. The test procedure with anti-D was easier and shorter than the remaining tests, but it can be only used for FMH assessment in RhD-negative mothers with RhD-positive newborns. In one clinical sample, it was impossible to distinguish fetal RBCs from maternal F cells in KBT and FC with anti-HbF but other tests were useful. CONCLUSION: In the four tests, correlation between expected and obtained results was appropriate (CCC Ì´1). Each test had some advantage and limitation in any clinical situation. Therefore, it is best to have opportunity to perform two or three assays in the laboratory.

Evaluation of passage of fetal erythrocytes into maternal circulation after invasive obstetric procedures.

AIM: The aim of this study was to evaluate the passage of fetal erythrocytes into the maternal circulation after invasive obstetric procedures, using the Kleihauer-Betke test, flow cytometry and α-fetoprotein concentration in maternal blood. MATERIAL AND METHODS: This was a prospective descriptive study on patients who underwent: amniocentesis, cordocentesis, chorionic villus sample, amniotic infusion, bladder drainage and ventricular-amniotic shunt to investigate the karyotype; treatment for hydrocephalus, oligohydramnios, obstructive uropathy and polyhydramnios; and investigation of lung maturity. Maternal blood samples were collected before and 60 min after the invasive obstetric procedure in order to evaluate the passage of fetal erythrocytes using the Kleihauer-Betke test, flow cytometry and α-fetoprotein concentration. RESULTS: In total, 43 invasive obstetric procedures were performed. The procedures performed were: 27 cases of amniocentesis (62.7%), seven cases of cordocentesis (16.2%), four chorionic villus samples (9.4%), two amniotic infusions (4.7%), two ventricular-amniotic shunts and one bladder drainage (2.3%). After one case of cordocentesis with two puncture attempts via the placenta, a significant increase in fetal erythrocytes was detected using the three methods. After another cordocentesis with one puncture via the placenta, a significant increase in fetal erythrocytes was detected using flow cytometry and α-fetoprotein concentration, but not through the Kleihauer-Betke test. The other 41 samples did not show any significant increase in fetal erythrocytes in the maternal blood. CONCLUSION: Invasive obstetric procedures performed during prenatal care are safe when performed by experienced professionals with the proper technique, with minimal chance of passage of fetal erythrocytes into the maternal compartment.

Two Cases of Severe Neonatal Anemia associated with Massive Feto-Maternal Hemorrhage / 대한주산의학회잡지

Feto-maternal hemorrhage, the presence of fetal red blood cells in the maternal circulation, occurs in up to 75% of pregnancies. But its volume is usually very small. Feto-maternal hemorrhage of more than 30 ml of whole blood is relatively rare. The key features that lead to early diagnosis are the maternal history, fetal monitoring, the clinical and laboratory findings of anemia and a negative Coombs' test. Diagnosis is confirmed by Kleihauer-Betke test. Perinatal problems include fetal distress, neonatal anemia, hypovolemic shock, and death. The fetal outcome depends on the amount and rate of bleeding. The initial hemoglobin level was a better predictor than the volume of bleeding. We present two cases of severe neonatal anemia associated with massive feto-maternal hemorrhage, which was confirmed by Kleihauer-Betke test.