Development of a new staining protocol for the Kleihauer-Betke test to facilitate the reading of difficult cases.

BACKGROUND: The Kleihauer-Betke (KB) test allows the detection of fetal red blood cells (containing fetal hemoglobin, HbF) in the maternal blood to identify and quantify potential fetal-maternal hemorrhages. In certain cases, detecting fetal red blood cells with conventional staining is difficult. False-positive results or overestimation of the quantity of fetal red blood cells may occur in cases of maternal hemoglobinopathy. In this study, we developed a new staining protocol to facilitate the reading of difficult smears and improve the precision of the quantification of fetal red blood cells; we also analyzed the performance of this new method. This study assessed blood samples with and without hemoglobin abnormalities, which present difficulties when interpreting the KB test. METHODS: The new staining formula is based on an improved elution technique and the use of a different stain instead of hematoxylin. To test this staining method, 16 samples from patients with abnormal hemoglobin electrophoresis and 14 samples from patients with normal hemoglobin electrophoresis were analyzed using the KB test with the classical staining method and the new staining method. In addition, a second series was prepared using the same samples spiked with fetal red blood cells from newborn blood, to compare the accuracy of the two methods in identifying fetal red blood cells. RESULTS: In the 60 slides analyzed with both staining methods, we found that the new technique improved the accuracy from 78 to 85%; lowered the coefficient of variation between the operators, which decreased from 20.7% to 12.7%; increased the specificity in our population from 56 to 70%; and decreased the number of false-positive cases by 30%. CONCLUSIONS: We successfully developed a new staining technique that facilitates the reading of difficult slides and improves the specificity of the detection of fetal red blood cells. This technique is recommended as a secondary method to use before sending the sample for additional exploration.

Spontaneous massive fetomaternal hemorrhage: two case reports and a literature review of placental pathology.

BACKGROUND: Massive fetomaternal hemorrhage (FMH) is a rare event during pregnancy that may cause severe fetal anemia or death. CASE PRESENTATION: This paper reports two cases of fetomaternal hemorrhage with unexplained reasons. Both cases required emergency caesarean sections for non-reassuring fetal status and were treated with neonatal blood transfusion. Fetomaternal hemorrhage was confirmed via maternal Kleihauer-Betke test. CONCLUSION: We found parenchymal pallor, increased nucleated red blood cells (nRBCs), and syncytial knots (SKs) in the placentas, which are compatible with fetal anemia. Immunohistochemical staining indicated VEGF, CD34, and CD31 expression in the endothelial cells of the capillaries, characteristic of massive FMH placenta. This article also reviews the particular histopathological changes in FHM placenta according to the placental lesion classification system.

Fetomaternal hemorrhage: Evidence from a multihospital healthcare system that up to 40% of severe cases are missed.

BACKGROUND: We previously reported fetomaternal hemorrhage (FMH) in 1/9160 births, and only one neonatal death from FMH among 219,853 births. Recent reports indicate FMH is not uncommon among stillbirths. Consequently, we speculated we were missing cases among early neonatal deaths. We began a new FMH initiative to determine the current incidence. METHODS: We analyzed births from 2011 to 2020 where FMH was diagnosed. We also evaluated potential cases among neonates receiving an emergent transfusion just after birth, whose mothers were not tested for FMH. RESULTS: Among 297,403 births, 1375 mothers were tested for FMH (1/216 births). Fourteen percent tested positive (1/1599 births). Of those, we found 25 with clinical and laboratory evidence of FMH adversely affecting the neonate. Twenty-one received one or more emergency transfusions on the day of birth; all but two lived. We found 17 others who received an emergency transfusion on the day of birth where FMH was not tested for, but was likely; eight of those died. The 42 severe (proven + probable) cases equate to 1/7081 births. We judged that 10 of the 42 had an acute FMH, and in the others it likely had more than a day before birth. CONCLUSIONS: We estimate that we fail to diagnose >40% of our severe FMH cases. Needed improvements include (1) education to request maternal FMH testing when neonates are born anemic, (2) education on false-negative FMH tests, and (3) improved FMH communications between neonatology, obstetrics, and blood bank.

Maternal ABO Discrepancy Caused by Massive Fetomaternal Hemorrhage: a Case Report.

BACKGROUND: Fetomaternal hemorrhage (FMH) can lead to severe, life-threatening fetal anemia depending on the amount of blood loss. FMH may be underdiagnosed as it is not routinely tested. In our report, we present a rare case of obvious mixed-field agglutination of maternal ABO forward typing caused by massive fetomaternal hemorrhage. METHODS: We retrospectively evaluated the clinical information of a 42-year-old pregnant woman who was admitted to our hospital at gestational week (GW) 36_5/7 due to fetal distress. She later delivered a male infant with severe anemia by cesarean section. RESULTS: This case had an unusual maternal hemoglobin elevation before delivery and the maternal blood type identification showed ABO discrepancy. After other causes were excluded, FMH was suspected. The Kleihauer-Betke (K-B) test was done on the mother's blood, indicating a massive FMH. CONCLUSIONS: Massive FMH may be a cause of ABO discrepancy of pregnant woman. FMH should be considered when we get a result of a mixed-field agglutination of pregnant woman with other possible causes excluded.

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.

Approach to red blood cell antibody testing during pregnancy: Answers to commonly asked questions.

OBJECTIVE: To provide family physicians with an understanding of blood bank tests performed during pregnancy. The value of routine blood type and antibody tests, as well as the follow-up required when a patient develops a red blood cell antibody or experiences a fetal-maternal hemorrhage (FMH) will be reviewed. SOURCES OF INFORMATION: The approach described is based on the authors' clinical expertise and peer-reviewed literature from 1967 to 2020. MAIN MESSAGE: An ABO and RhD group and antibody screen test is performed on every pregnant patient during the first trimester. Although antibodies to red blood cell antigens occur infrequently, some can lead to substantial adverse fetal or neonatal consequences including hemolytic disease of the fetus and newborn. Early identification and quantification of important antibodies ensures that at-risk mothers are referred to and followed by obstetricians experienced with high-risk care. Another valuable and related test is the FMH test. For RhD-negative women, these tests are performed at every delivery and following antepartum events that could contribute to FMH. This test determines the number of fetal red blood cells in the maternal circulation and is used to determine the dose of Rh immune globulin an RhD-negative mother requires to prevent alloimmunization to fetal RhD. CONCLUSION: An understanding of blood bank tests performed during pregnancy and their role and limitations is vital to optimal practice and aids clinicians in their decision making. When there is doubt or confusion regarding antenatal testing or immunoprophylaxis, consult the regional laboratory or transfusion medicine specialists for additional guidance.

Fetomaternal Hemorrhage: Can We Use Hemoglobin Electrophoresis as a Diagnostic Tool?

Fetomaternal hemorrhage (FMH) is usually detected by either the Kleihauer-Betke (KB) test or by 2 cytometry, the latter of which represents the gold standard of FMH diagnosis today. But what do we do when neither method is available? We present two cases of suspected FMH due to their characteristic signs and symptoms that were ultimately confirmed by hemoglobin electrophoresis (HE).

Intermittent Sinusoidal Fetal Heart Rate and Massive Maternal-Fetal Hemorrhage: A Case Report.

BACKGROUND: A sinusoidal fetal heart rate pattern is a well-documented sign of maternal-fetal hemorrhage; however, an intermittent sinusoidal pattern is rare. CASE: This is a case of a 26-year-old primigravida who presented with decreased fetal movement. On fetal heart rate monitoring there were episodes of intermittent sinusoidal fetal heart rate pattern, and the baby was born with anemia. CONCLUSION: This case highlights the need for increased vigilance and consideration of a diagnosis of maternal-fetal hemorrhage when an intermittent sinusoidal fetal heart rate pattern is identified.

Case Report of Anemia Following Fetal-Maternal Hemorrhage.

BACKGROUND: Any maternal history of blood loss, ABO or Rh incompatibility, and hydrops fetalis often leads to suspicion of neonatal anemia postnatally. When maternal history consists only of decreased fetal movement, recognition of neonatal anemia can be problematic. CLINICAL FINDINGS: This case was a transported late preterm neonate who presented initially with persistent hypoxia unresponsive to usual respiratory support. On examination, mild paleness was noted. PRIMARY DIAGNOSIS: Anemia caused by fetal-maternal hemorrhage was the ultimate diagnosis confirmed by a Kleihauer-Betke test on maternal serum examining fetal cells. INTERVENTIONS: Neonatal resuscitation included positive pressure ventilation, oxygen, and intubation. However, oxygenation did not improve prompting consultation with the neonatologist. Sedation and a paralytic were given. A chest radiograph ruled out pneumothoraces and pleural effusions as causative. Initiation of inhaled nitric oxide produced a mild response. Eventually, the transport nurse obtained a complete blood count indicating severe anemia, which prompted an emergent blood transfusion. The accepting neonatology team consulted with the obstetrician and a Kleihauer-Betke test was performed on mother's blood confirming a large fetal-maternal hemorrhage. OUTCOMES: This neonate responded well to blood transfusions, a pressor, and respiratory support and was discharged home at 7 days of life. PRACTICE RECOMMENDATIONS: Recognition of postnatal anemia is vital to sustaining life and this can occur in the transport environment. When maternal history is nonspecific and a neonate is hypoxic, uncommon causes of hypoxia can be identified with consultation and a complete blood count.

The Recurrence Risk of Fetomaternal Hemorrhage.

Massive fetomaternal hemorrhage (FMH) can cause devastating pregnancy outcomes. Perinatal prognosis may be improved by intrauterine transfusion, but the appropriate management for these pregnancies remains unclear. To determine the recurrence risk of FMH after intrauterine transfusion, we performed a systematic review of all case reports/series of patients with proven FMH treated with intrauterine transfusion and who had subsequent follow-up of at least 72 h until delivery. This revealed 13 cases, with 1 additional case from our institution. Ten patients (71.4%) had a second episode of FMH requiring a second intrauterine transfusion. Five patients (35.7%) required at least 3 intrauterine transfusions. The time interval between intrauterine transfusions was progressively reduced. The gestational age at the onset of signs/symptoms was 26.6 ± 2.1 weeks, and gestational age at delivery was 34.2 ± 4.2 weeks. Two cases of fetal demise (14.3%) and no neonatal deaths were recorded. Limited postnatal follow-up on 8 neonates was normal. The mean neonatal hemoglobin and transfusion rates were 13.2 ± 5.7 g/dL and 33.3%, respectively. Close fetal monitoring, likely daily, is necessary to recognize FMH recurrence. Several transfusions may be necessary once FMH is diagnosed if pregnancy is allowed to continue > 72 h.

Evaluation of stillbirth.

PURPOSE OF REVIEW: To review current evidence regarding the most useful tests for evaluation of potential causes of stillbirth. RECENT FINDINGS: Stillbirth remains one of the most devastating obstetric complications. Recent advances include work investigating the highest yield diagnostic tests for determining a cause of death in stillbirths. Placental pathology and fetal autopsy improve the diagnostic yield when combined with maternal clinical history. Additional tools include genetic evaluation, and testing for antiphospholipid antibodies and fetal-maternal hemorrhage based on the clinical scenario. However, routine testing for heritable thrombophilias or infection has not proven to be useful. In cases in which fetal autopsy is not acceptable to patients or possible for logistical reasons, alternative forms of fetal evaluation should be considered. SUMMARY: A systematic approach to stillbirth evaluation is useful in identifying a potential cause of death. This should incorporate clinical information as well as placental pathology and fetal autopsy. Based on clinical characteristics further testing may be indicated.

Untimely diagnosis of fetomaternal hemorrhage: what went wrong?

Fetomaternal hemorrhage (FMH) is an obstetrical challenge. It is defined as a passage of fetal blood into the maternal circulation or vice versa, which might complicate pregnancy or delivery. Most cases of acute and chronic FMH are idiopathic in origin and involve uncomplicated near-term pregnancies. Yet, due to the lack of universal screening, heterogeneous clinical presentation and insufficient clinicians awareness, in some cases FMH may present as immediate fetal compromise or even stillbirth as the most devastating consequence. We made a review of the literature of the FMH clinical cases of fetal/neonatal death in order to focus on the available diagnostic tools and their limitations. Cardiotocography, biophysical profile, middle cerebral artery peak systolic volume and current laboratory tests were studied and evaluated as diagnostic tools for FMH. International guidelines are needed to help clinicians make a prompt identification of FMH. Moreover, a standardized management protocol is essential in order to improve fetal-neonatal outcomes.

Should optimal timing between two intrauterine transfusions be based on estimated daily decrease of hemoglobin or on measurement of fetal middle cerebral artery peak systolic velocity?

BACKGROUND: To best predict the recurrence of fetal anemia after intrauterine transfusion (IUT), the measurement of middle cerebral artery peak systolic velocity (PSV) and the estimation of hemoglobin (Hb) daily decrease are compared. STUDY DESIGN AND METHODS: A retrospective study including 38 patients who had at least two IUTs in a context of red blood cell alloimmunization was conducted. PSV values before first, second, and third IUTs were collected and expected Hb level was calculated according to various Hb daily decrease formulas as proposed in the literature. RESULTS: Comparison of PSV receiver operating characteristic curves with the various Hb levels did not find any significant difference between first and second IUTs. On the other hand, we found a significant difference between the second and third IUTs, with better prediction of fetal anemia through Hb decrease calculation, whatever the formula. Between the second and third IUTs, no formula was significantly better than the others. CONCLUSION: The timing of a second transfusion can be difficult to determine with certainty, but PSV can give an accurate assessment of when to resample the fetus with probably a higher recommended threshold for the diagnosis of fetal anemia. Subsequent to a second transfusion, the intertransfusion interval should be based on estimated Hb decrease rather than PSV thresholds, whatever the chosen formula proposed in the literature. Larger numbers are needed to definitely make this recommendation and it will be interesting to evaluate correlation between different antibodies.

Biochemical analysis of intraplacental choriocarcinoma and fetomaternal transfusion.

Intraplacental choriocarcinoma is one of the rarest forms of gestational tumors and is believed to be one of the causes of fetomaternal transfusion (FMT). A 35-year-old woman, gravida 2, para 2, with a history of two vaginal deliveries, was incidentally diagnosed as having stage I gestational intraplacental choriocarcinoma with a FIGO/World Health Organization 2000 risk score of 2 after term delivery. This disease caused neonatal anemia but did not metastasize to either the mother or infant. Short tandem repeat analysis with laser microdissection revealed that the tumor had originated from the current pregnancy. Serological test and immunohistochemical analysis revealed that the patient and her baby suffered from FMT. She has been free from disease without any medical intervention for the last 1 year. A combination of multiple biochemical analyses might help us diagnose the precursor pregnancy of a gestational choriocarcinoma and FMT.

Fetomaternal hemorrhage complicated pregnancy: risks, identification, and management.

PURPOSE OF REVIEW: This article aims not only to review recent literature about the clinical features of massive fetomaternal hemorrhage (FMH) and identification of risk factors, but also to alert obstetricians and pediatricians to this underdiagnosed and underestimated severe obstetrical issue. In addition, a simplified flow chart for the antenatal management of suspected FMH is proposed. RECENT FINDINGS: Improvements in obstetrical and neonatal care have decreased perinatal morbidity and mortality and the rate of stillbirth. Unfortunately, because of the nonspecific signs on presentation, adverse outcome associated with massive FMH has not followed this trend and still has devastating consequences. As even the definition varies among publications and there is lack of universal screening, the real nature still remains obscure. Improvements in the diagnosis of fetal anemia, laboratory and intrauterine transfusion techniques, and the implementation of prenatal and postnatal neuroprotection give some hope for the better outcome in the most severe cases. Unfortunately, obstetricians' awareness of the massive FMH remains still at an unacceptably low level. SUMMARY: There is an urgent need for the internationally accepted definition, standardized pregnancy management protocol, and structured follow-up of neonates from such pregnancies. We suggest the international registry of massive FMH cases.

Flow Cytometry in Detection of Fetal Red Blood Cells and Maternal F Cells to Identify Fetomaternal Hemorrhage.

Accurate detection and quantitation of fetomaternal hemorrhage (FMH) is critical to the obstetric management of rhesus D alloimmunization in Rh-negative pregnant women. The flow cytometry is based on the detection of fetal red blood cells using a monoclonal anti-HbF antibody, and is the method most indicated for this estimation. The objective of this study was to quantify fetal red blood cell levels of pregnant women using flow cytometry. We analyzed 101 peripheral blood samples from Rh-negative and Rh-positive women, whose mean age was 24 years (20-32 years), after vaginal delivery or cesarean section. Our study showed that 53% of pregnant women had fetal red blood cells levels <2.0 mL, 31% between 2.0-3.9 mL, 16% between 4.0-15.0 mL, and 1% >15.0 mL. Accurate quantitation of fetal red blood cells is necessary to determine the appropriate dose of anti-D (RHD) immunoglobulin to be administered to pregnant or postpartum women.

Two cases of asymptomatic massive fetomaternal hemorrhage.

Evaluation of fetomaternal hemorrhage (FMH) in the immediate postpartum period is critical for the timely administration of Rh immunoglobulin (RhIG) prophylaxis to minimize the risk of alloimmunization in D-negative mothers of D-positive newborns. We report a series of two clinically-unsuspected cases of massive FMHs identified at our university medical center. Retrospective records of two cases of massive FMH were investigated using the electronic medical record. After positive fetal bleed screens, flow cytometric analysis for hemoglobin F was performed to quantify the volume of the hemorrhages in both cases. Flow cytometric enumeration with anti-D was also performed in one case. The two patients had 209.5 and 75 mL of fetal blood in circulation, resulting in 8 and 4 doses of RhIG administered, respectively. For the former patient, flow cytometric analysis with anti-D ruled out hereditary persistence of fetal hemoglobin and supported the fetal origin of the red cells. Due to the clinically-silent nature of both hemorrhages, further evaluation of the newborns' blood was not performed. These cases highlight the importance of rapidly obtaining accurate measurements of fetal blood loss via flow cytometric analysis in cases of FMH, particularly in clinically-unsuspected cases, to ensure timely administration of adequate immunoprophylaxis to D-negative mothers.

Fetomaternal hemorrhage (FMH), an update: review of literature and an illustrative case.

BACKGROUND: Blood trafficking from fetus to mother and vice versa is a well-known physiological event that occurs at any stage in pregnancy. If the fetus looses high blood quantities to the maternal blood stream it becomes symptomatic. These symptoms can vary from cardiovascular distress to fetal death. MATERIALS AND METHODS: We give a review of current literature on Fetomaternal hemorrhage (FMH). CONCLUSION: This article highlights the importance of physician's awareness on detecting this rare but life threatening entity with both severe consequences for mother and neonate. The traditional measurement of FMH and the co-usage of alpha-fetoprotein are debated. To conclude we describe and discuss an illustrative case of FMH. This article gives an applicatory overview of symptoms, diagnostics and treatment of FMH to facilitate physicians to detect this disease precociously.

[Standardization of the quantitative flow cytometric test with anti-D antibodies for fetomaternal hemorrhage in RhD negative women]. / Standaryzacja metody ilosciowej oceny przecieku plodowo-matczynego u kobiet RhD ujemnych przy pomocy cytometrii przeplywowej z uzyciem przeciwcial anty-D.

BACKGROUND: In order to determine the appropriate dose of anti-D immunoglobulin to be administered as a preventive measure against hemolytic disease of the fetus/newborn in the subsequent pregnancy it is necessary to assess the number of fetal red blood cells that infiltrate/penetrate into the maternal circulation as a result of fetomaternal hemorrhage (FMH). One of the quantitative methods of FMH analysis is based on flow cytometry (FACS) which makes use of monoclonal antibodies to RhD antigen (anti-D test). The aim of the study was to further develop the method, evaluate its sensitivity and reproducibility and to compare it with the test based on the detection of fetal hemoglobin (HbF). MATERIAL AND METHODS: The FACS study involved 20 RhD negative pregnant women and 80 RhD negative women after delivery. The following monoclonal antibodies were used: BRAD 3 FITC (anti-RhD antigen), CD45 PerCP (anti leukocyte antigen CD45), and anti-HbF PE. RESULTS: The fluorescence intensity of cells incubated with BRAD 3 FITC was demonstrated to depend on the RhD antigen expression, though the anti-D test also detects the weak D variant. The CD45 PerCP antibodies increased the sensitivity of anti-D test since they eliminated the leukocytes which non-specifically bind anti-D from the analysis. The presence of anti-D antibodies in maternal plasma does not affect the quantitative assessment of the fetal RhD positive fetal cells with BRAD 3 FITC. In case of FMH, the results of the anti-D test were similar to those with anti-HbF antibodies. CONCLUSIONS: The flow cytometric test with anti-D and anti-CD45 is useful in the assessment of the fetomaternal hemorrhage in RhD negative women. The sensitivity of the test is estimated at 0.05%.