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.

Massive fetomaternal haemorrhage warranting novel use of tandem isovolumetric partial exchange transfusion and therapeutic hypothermia.

A newborn male infant was pale, hypotonic, and had respiratory distress after delivery. Venous cord blood gas revealed a severe metabolic acidosis. His initial examination was consistent with moderate encephalopathy and laboratory testing uncovered severe congenital anaemia (haematocrit 0.127 L/L). He met the clinical criteria for therapeutic hypothermia (TH) and required red blood cell transfusions, but due to the severity of his anaemia, an exchange transfusion was favoured to prevent transfusion-associated circulatory overload. There are no previous reports of these procedures completed in tandem, but the benefits were perceived to outweigh the risks. During the 72 hours of TH, the infant received an isovolumetric partial exchange transfusion and tolerated both treatments without any adverse clinical events.Kleihauer-Betke testing detected a massive chronic fetomaternal haemorrhage with 475 mL (164 mL/kg) of blood. A brain MRI completed prior to discharge was normal. At 6 months of age, he is growing and developing normally.

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.

Massive fetomaternal hemorrhage in a monochorionic-diamniotic twin pregnancy: a case report.

Fetomaternal hemorrhage (FMH) result into severe, life-threatening fetal anemia and cause intrauterine death of the fetus. It is tough for an early diagnosis of FMH before pregnancy and few authors reported FMH in a twin pregnancy. Therefore, we reported a case of massive FMH. The patient felt a decrease in fetal movements at 33+5 gestational weeks. Cardiotocography showed sinusoidal heart rate patterns in one fetus. The fetal hemoglobin level in maternal blood was 6.4% (normal range for single pregnancy, 0.0%-2.0%). Since the patient was diagnosed with fetal distress, cesarean section was performed and both babies delivered to receive neonatal treatment. Severe anemia was apparent in both neonates, based on red blood cell count, hemoglobin concentration, and hematocrit of 0.75 × 1012/L and 0.61 × 1012/L, 2.8 g/dL and 2.4 g/dL, and 10.0% and 8.4%, respectively. The neonates were admitted to the intensive care unit for prematurity care and presently are well. In our experience, an early diagnosis of FMH contributed to saving fetus. Obstetricians should highlight fetal movements counting to every patient. Once massive FMH occurs in monochorionic twins, both fetuses may develop severe anemia and require emergency intervention.

Management and Follow-up of Massive Fetomaternal Hemorrhage Requiring High-Dose Rh Immune Globulin: A Case Report.

OBJECTIVES: Massive fetomaternal hemorrhage (FMH) is rare and reported to be the cause in approximately 3% of all fetal deaths. Maternal management of massive FMH includes prevention of Rh(D) alloimmunization in Rh(D)-negative mothers by administration of Rh(D) immune globulin (RhIG). METHODS: We describe a case of a 30-year-old O-negative, primigravida woman who presented at 38 weeks of gestation with decreased fetal movements. She underwent an emergency cesarean section and delivered an O-positive baby girl who died shortly after birth. RESULTS: The patient's FMH screen was positive, with a Kleihauer-Betke test demonstrating 10.7% fetal blood in maternal circulation. The calculated dose of 6,300 µg RhIG was given prior to discharge over 2 days using an intravenous (IV) preparation. Antibody screening a week after discharge showed anti-D and anti-C. The anti-C was attributed to acquired passive immunity from the large dose of RhIG. Anti-C reactivity waned and was negative at 6 months, but the anti-D pattern persisted at 9 months postdelivery. Negative antibody screens were noted at 12 and 14 months. CONCLUSIONS: This case highlights the immunohematology challenges of IV RhIG as well as the success in preventing alloimmunization with IV RhIG given the patient's complete resolution of anti-C and no anti-D formation, with a subsequent healthy pregnancy.

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.

Fetomaternal hemorrhage: a clue to intraplacental choriocarcinoma and neonatal malignancy.

Fetomaternal hemorrhage (FMH) is a known cause of neonatal anemia due to fetal blood loss to the maternal circulation, occurring when the maternal-fetal barrier is disrupted. Several causes must be considered, although in most cases the etiology remains unknown. Intraplacental choriocarcinoma (ICC) is a rare entity and has been related with massive FMH, intrauterine fetal demise, severe neonatal anemia and metastatic choriocarcinoma in both mother and infant. There are 25 cases of histopathologically confirmed ICC complicated with FMH described in the literature. Because FMH occurs unexpectedly and the majority of patients with ICC are asymptomatic, this diagnosis may be missed. Once FMH is confirmed, underlying malignancy should be kept in mind. The authors present a case report of severe neonatal anemia following FMH related to ICC and highlight the importance of serum ß-hCG monitoring in cases of massive FMH.

Diagnostic accuracy of Kleihauer-Betke (Kb) testing to predict fetal outcomes associated with fetomaternal hemorrhage: a retrospective cohort study.

OBJECTIVE: To evaluate the diagnostic and screening utility of Kleihauer-Betke (KB) testing as a triage tool in predicting adverse fetal outcomes associated with fetomaternal hemorrhage (FMH). STUDY DESIGN: Single center retrospective cohort study evaluated a primary composite outcome of fetal complications associated with FMH between KB-negative and KB-positive test groups. Screening tests for sensitivity, specificity, positive predictive value and negative predictive value were determined. RESULTS: 641 women (97%) had KB-negative and 22 (3%) had KB-positive tests. The primary composite outcome between KB-negative and KB-positive pregnancies was similar (30% vs. 36%, p = 0.54). Screening exhibited high specificity (97%), however, test sensitivity was poor (4%) with only moderate positive and negative predictive values (36.4 and 69.7%). CONCLUSION: Fetal outcomes associated with FMH were not significantly different between KB-positive and KB-negative test cohorts; KB testing offers no diagnostic precision in the emergency triage evaluation of women with suspected FMH.

Severe neonatal anemia affected by massive fetomaternal hemorrhage: a single-center retrospective observational study.

OBJECTIVES: Massive fetomaternal hemorrhage (FMH) is a rare but sometimes life-threatening event, and surviving neonates may suffer major neurological complications. Severe neonatal anemia (SNA) affected by massive FMH is less reported in the literature. This study aims to explore the clinical characteristics, laboratory diagnoses, treatments and outcomes of SNA affected by massive FMH. METHODS: Data were collected retrospectively from the hospital's electronic medical record system. All neonates born in the hospital and admitted to the neonatal unit diagnosed as SNA affected by massive FMH from 1 January 2013 to 31 June 2017 were included. RESULTS: A total of 8 cases of SNA affected by FMH were identified among 6825 neonates admitted to the neonatal unit. They all presented with pallor but without hydrops at birth. Median gestational age and birthweight were 375/7 (360/7‒401/7) weeks and 2,625 (2300‒3050) g, respectively. Median hemoglobin level was 39.5 (25‒53) g/L at birth and 109.5 (94-127) g/L at discharge. Median maternal serum alpha-fetoprotein (AFP) was 3958.5 (1606‒14,330) ng/mL, which was significantly increased. Three out of eight cases manifested as antenatal decreased fetal movement. Only 1 with the lowest initial hemoglobin 25 g/L manifested as characteristic sinusoidal fetal heart rate tracing and suffered severe neonatal asphyxia and hypovolemic shock. Having experienced resuscitation, he was admitted to the neonatal unit and received twice transfusion of cross-matched red blood cells there. Another case with the initial hemoglobin 45 g/L received positive pressure ventilation and once transfusion. All cases were successfully discharged with a median hospital stay of 8 (5-12) days. Follow-up was available for 6 (75%) of 8 neonates (age range 13 months to 50 months), and all infants were observed to be in good condition with normal neurological status. In our series of eight cases, there were no neonatal deaths. CONCLUSION: This study strengthens the idea that maternal AFP testing is valuable to confirm massive fetomaternal hemorrhage. Surviving neonates of massive FMH might have a good outcome despite severe anemia at birth.

Reduced fetal movements: the case of fetomaternal hemorrhage. Case series and proposal of a management protocol.

Fetomaternal hemorrhage (FMH) was reported more than 60 years ago for the first time defined by the transfer or transfusion of fetal blood into the maternal circulation before or during delivery. The transfused volume is usually very small but when this value exceeds, it may be clinically significant. Antenatal diagnosis of severe FMH is difficult and it can be suspected in case of reduction of fetal movements, abnormal cardiotocography and ultrasound. FMH is associated to different adverse outcomes and admission to neonatal intensive care. The low incidence of FMH limits the studies, thus being able to rely only on diagnosis and retrospective studies. We present case series of FMH and analyze the steps with the purpose of defining a flow-chart for early diagnosis and management of FMH.

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.

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.

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.

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.

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).