Massive antenatal fetomaternal hemorrhage: evidence for long-term survival of fetal red blood cells

BACKGROUND: Massive fetomaternal hemorrhage (FMH) can lead to life-threatening anemia. Quantification based on flow cytometry with anti-hemoglobin F (HbF) is applicable in all cases but underestimation of large fetal bleeds has been reported. A large FMH from an ABO-compatible fetus allows an estimation of the life span of fetal red blood cells (RBCs) in the maternal circulation. CASE REPORT: The mother went to the obstetrician twice antepartum owing to symptoms assumed to be preeclampsia; that, however, was not found. She later delivered by cesarean section owing to diminished fetal movements. No fetal weight gain was observed during the last 2 weeks of pregnancy. STUDY DESIGN AND METHODS: Fetal RBCs were quantified by flow cytometry with anti-HbF, anti-Fy(a), anti-s, and anti-Jk(b) on a regular basis. RESULTS: The infant had anemia at delivery and an FMH was determined to be 314 +/- 17 mL (+/- SD) of whole blood. It is assumed that the two antenatal visits were associated with the FMH. Postpartum follow-up showed that fetal RBCs in the maternal circulation were detectable with anti-HbF up to 119 days. Quantification by flow cytometry based on anti-HbF was in agreement with quantification based on anti-Fy(a), anti-s, and anti-Jk(b), although they were less sensitive. CONCLUSION: ABO-compatible fetal RBCs from an FMH had a life span in the maternal circulation close to that of adult RBCs.     

Massive fetomaternal hemorrhage: clearance of fetal red blood cells after intravenous anti-D prophylaxis monitored by flow cytometry

BACKGROUND: The clearance of D+ red blood cells (RBCs) from the circulation in D- individuals mediated by passively administered anti-D occurs by opsonization with the antibody and subsequent removal in the spleen. Few data exist on the kinetics of clearance of large volumes of D+ RBCs from the maternal circulation by anti-D in clinical cases of massive fetomaternal hemorrhage (FMH). CASE REPORT: A 33-year-old D- woman delivered a D+ female infant by emergency cesarean section for suspected fetal anemia. A massive FMH, initially estimated to be approximately 142 mL of RBCs, was found. In addition to the standard dose of intramuscular (IM) anti-D (300 microg) given immediately after delivery, 2700 microg of anti-D was administered intravenously (IV). The clearance of D+ fetal cells from the maternal circulation was monitored by flow cytometry in samples obtained on a daily basis using anti-D. The mother had no detectable anti-D 6 months after delivery. RESULTS: No clearance of fetal cells was apparent after the insufficient dose of IM anti-D. The IV administration of anti-D caused accelerated clearance of D+ fetal RBCs with a t1/2 of 24.5 hours. D+ reticulocytes comprised 4.2 percent of all D+ cells in the maternal circulation at delivery suggesting acute fetal blood loss. CONCLUSIONS: The approach used in this report allowed a detailed analysis of the kinetics related to the clearance of fetal D+ RBCs. Simultaneous measurements of fetal reticulocytes and fetal RBCs in maternal blood may establish the timing of an FMH.     

Comparison of haemoglobin F detection by the acid elution test, flow cytometry and high-performance liquid chromatography in maternal blood samples analysed for fetomaternal haemorrhage

Background and Objective: All pregnant women undergoing blood grouping at Southmead Hospital are offered haemoglobinopathy screening by high‐performance liquid chromatography (HPLC). RhD‐negative women who deliver RhD‐positive infants are tested for fetomaternal haemorrhage (FMH) by acid elution (AE). The effectiveness of these two assays for quantitation of FMH was compared with flow cytometry (FC). Materials and Methods: The relationship between expression of haemoglobin F (HbF) in individual cells by AE and FC and quantitation of HbF in haemolysates by HPLC was investigated, using maternal samples with unusually high levels of HbF‐positive maternal cells (F cells) or with large FMH (fetal cells). Standard anti‐D FC was performed to quantitate fetal D‐positive cells in D‐negative women and compared with FMH estimated by AE and HbF FC. Results: AE overestimated FMH when maternal F cells were increased. HbF FC distinguished F cells from fetal cells. Values of HbF determined by HPLC were less than the level of 5% used for investigation of raised fetal haemoglobin, even in the maternal samples with elevated F cells or massive FMH. Conclusions: To quantitate FMH, measurement of HbF using FC was more sensitive and accurate than AE or HPLC. HbF FC is the method of choice when results from routine investigation using AE or standard anti‐D FC are discrepant or when there is maternal and fetal RhD compatibility.     

Testing for hematologic disorders and complications

This review summarizes state-of-the-art and emerging techniques in the antenatal diagnosis of fetal anemia and hemoglobinopathies. Fetal anemia may result from hemolytic disease, hemorrhage, suppression of erythropoiesis, infection (eg, parvovirus B19), or trauma. The clinical laboratory plays an essential role in the evaluation of these disorders by way of the use of various hematologic, biochemical, serologic, cytometric, and molecular genetics methods. Hemoglobinopathies are the most common class of single gene disorders worldwide. The authors have used the example of homozygous alpha-thalassemia major (Hb Barts disease) as a paradigmatic case for antenatal hemoglobinopathy screening. Perhaps the most familiar indication for hematologic screening in pregnancy is HDFN, most commonly in pregnancies previously sensitized to the RhD antigen. All pregnant women, regardless of their past medical or obstetric history or previous antibody screens, should have ABO/Rh blood typing and a red cell antibody screen performed at the first prenatal visit. Long-established methods for assaying FMH (KB method), microcytosis (hemogram with red cell indices), and blood group incompatibility (direct antigen test, serologies) remain critical for rapid, sensitive diagnosis. Analysis of fetal free DNA in maternal plasma holds the promise for rapid, ultrasensitive, and noninvasive detection of many fetal hematologic disorders.     

Overestimation of fetomaternal haemorrhage by the acid-elution technique in mothers with β-thalassaemia minor

Summary. In Rh-negative women, it is important to quantify the magnitude of an Rh-positive fetomaternal haemorrhage (FMH) so that sufficient Rh immune globulin (RhIg) can be administered early in the postpartum period to prevent alloimmunization. The standard post-partum dose of Rhlg varies from 100 μg in the UK to 300 μg in North America. It is therefore important to identify all Rh-negative women who have had an FMH greater than 10 ml in the UK or greater than 30 ml in North America because an FMH greater than these amounts will affect the dose of RhIg that is administered. As acid-elution techniques can overestimate the magnitude of an FMH in the presence of an elevated maternal haemoglobin F level, we performed a prospective study to determine how often this occurred. Of 1,894 consecutive Rh-negative mothers who delivered Rh-positive infants, whose blood was screened for an FMH greater than 10 ml of fetal blood using an acid-elution procedure, 11 were found to have an FMH over 10 ml. In five of these 11 women, the volume of FMH was less than 10 ml using an alternative technique (rosette test) to assess the FMH size. Six of these women were found to have β-thalassaemia minor on the basis of a low MCV, and high haemoglobin A₂ and/or high haemoglobin F levels. In five of these the FMH was significantly overestimated by the acid-elution technique compared to the rosette technique. Therefore, in the presence of a maternal condition, which may result in an elevated haemoglobin F level, an FMH estimated to be over 10 ml in the UK or 30 ml in North America using an acid-elution procedure, should be confirmed by an alternative technique, which does not involve the estimation, directly or indirectly, of haemoglobin F.     

Non-immune hydrops fetalis: Two case reports

BACKGROUNDFetal hydrops is a serious condition difficult to manage, often with a poor prognosis, and it is characterized by the collection of fluid in the extravascular compartments. Before 1968, the most frequent cause was the maternal-fetal Rh incompatibility. Today, 90% of the cases are non-immune hydrops fetalis. Multiple fetal anatomic and functional disorders can cause non-immune hydrops fetalis and the pathogenesis is incompletely understood. Etiology varies from viral infections to heart disease, chromosomal abnormalities, hematological and autoimmune causes.CASE SUMMARYA 38-year-old pregnant woman has neck lymphoadenomegaly, fever, cough, tonsillar plaques at 14 wk of amenorrhea and a rash with widespread itching. At 27.5 wk a fetal ultrasound shows signs of severe anemia and hydrops. Cordocentesis is performed with confirmation of severe fetal anemia and subsequent fetal transfusion. The karyotype is 46, XX, array-comparative genome hybridization (CGH) negative, and infectious tests are not conclusive. In the following days there is a progressive improvement of the indirect signs of fetal anemia. At 33.6 wk, for relapse of severe fetal anemia, further fetal transfusions are necessary and an urgent cesarean section is performed. On the day 12 of life, for the detection of anemia, the newborn is subjected to transfusion of concentrated red blood cells and begins treatment with erythropoietin. Later there is a normalization of blood chemistry values and the baby does not need new transfusions. A 29-year-old pregnant woman, with Sjogren''s syndrome and positive Anti-Ro/SSA antibodies, is subjected to serial fetal ecocardio for branch block. At 26.5 wk there is a finding of fetal ascites. Infectious disease tests on amniotic fluid are negative as well as quantitative fluorescent polymerase chain reaction, Array CGH. At cordocentesis Hb is 1.3 mmol/L, consequently fetal transfusion is performed. Also in this case, due to continuous episodes of relapse of fetal anemia with consequent transfusions, at 29.4 wk a cesarean section is performed. On day 9 of life, a treatment with erythropoietin is started in the newborn, but the baby needs three blood transfusions. The search for autoantibodies in the baby found SS-A Ro60 positive, SSA-Ro52 positive and SS-B negative. The hemoglobin values normalized after the disappearance of maternal autoantibodies.CONCLUSIONAn attempt to determine the etiology of hydrops should be made at the time of diagnosis because the goal is to treat underlying cause, whenever possible. Even if the infectious examinations are not conclusive, but the pregnancy history is strongly suggestive of infection as in the first case, the infectious etiology must not be excluded. In the second case, instead, transplacental passage of maternal autoantibodies caused hydrops fetalis and severe anemia. Finally, obstetric management must be aimed at fetal support up to an optimal timing for delivery by evaluating risks and benefits to increase the chances of survival without sequelae.     

Fetomaternal hemorrhage: incidence, risk factors, time of occurrence, and clinical effects

Most women have only very small amounts of fetal blood in their circulations following pregnancy and delivery: the volume is less than 0.5 mL of whole blood in 93 percent of women, less than 1 mL in 96 percent, and less than 2 mL in 98 percent. FMH of 30 mL or more occurs in just 3 of 1000 women. When the FMH was 150 mL or more, 15 of 41 infants did not survive Rh-negative women with FMH of more than 30 mL of Rh-positive whole blood are at increased risk of Rh immunization, and thus the outcome of their future pregnancies also may be affected. ABO-compatible fetal red cells that have entered the maternal circulation have a life span similar to that of adult cells. ABO-incompatible fetal red cells may be cleared rapidly, but in some cases they circulate for weeks. Most FMHs of 30 mL or more occur before labor, delivery, or cesarean section. The majority occur with minimal clinical signs and symptoms in apparently normal pregnancies. The identification of postpartum Rh-negative women who have 30 mL or more of Rh-positive fetal blood in their circulation is important so that sufficient RhIG for immune suppression can be administered. It appears that more than one-half of women with FMH of 30 mL or more would not be identified if protocols were adopted to test only women in pregnancies considered to be at high risk.     

流式细胞术非侵入性检测胎儿血型的研究

目的　建立流式细胞术非侵入性检测胎儿ABO、Rh(D)血型的实验方法。方法　采用吸收放散试验 ,从人源血清中获得IgG抗 A、抗 B试剂。依据胎儿父母的血型 ,选择IgG的抗 A、抗 B和抗 D作一抗与孕妇外周血红细胞反应 ,以羊抗人IgGF(ab′) 2 FITC作二抗连接A、B、D抗体 ,同时用抗 i PE标记孕妇外周血中的胎儿红细胞。依据所获荧光标记 (FITC和PE)的孕妇和胎儿细胞的散点图格局 ,分析胎儿细胞的ABO和Rh血型。结果　应用所研究的流式细胞检测技术 ,对 6 9名妊娠 8～ 39周的孕妇检测 ,13名孕妇外周血中因未检出胎儿红细胞而无法鉴定 ,其余 5 6名孕妇均检出胎儿细胞 ,她们的胎儿血型产前检测结果与胎儿出生后的检测结果完全吻合。结论　流式细胞术方法能在产前准确和无损伤地检测胎儿血型 ,可用于怀孕期间的新生儿溶血病的诊断和预防工作     

Direct antiglobulin test-negative autoimmune hemolytic anemia in a patient with β-thalassemia minor during pregnancy: A case report

BACKGROUNDSevere refractory anemia during pregnancy can cause serious maternal and fetal complications. If the cause cannot be identified in time and accurately, blind symptomatic support treatment may cause serious economic burden. Thalassemia minor pregnancy is commonly considered uneventful, and the condition of anemia rarely progresses during pregnancy. Autoimmune hemolytic anemia (AIHA) is rare during pregnancy with no exact incidence available. CASE SUMMARYWe report the case of a 30-year-old β-thalassemia minor multiparous patient experiencing severe refractory anemia throughout pregnancy. We monitored the patient closely, carried out a full differential diagnosis, made a diagnosis of direct antiglobulin test-negative AIHA, and treated her with prednisone and intravenous immunoglobulin. The patient gave birth to a healthy full-term baby.CONCLUSIONCoombs-negative AIHA should be suspected in cases of severe hemolytic anemia in pregnant patients with and without other hematological diseases.     

Fetomaternal hemorrhage in normal vaginal delivery and in delivery by cesarean section

BACKGROUND: The objective was to determine the incidence and volume of fetomaternal hemorrhage (FMH) in normal vaginal delivery and in delivery by cesarean section. Determination of these variables would enable optimalization of guidelines for D alloimmunization prophylaxis. STUDY DESIGN AND METHODS: In a prospective cohort study, a total of 3457 examinations were performed, 2413 after normal vaginal delivery and 1044 after cesarean delivery. FMH was assessed by flow cytometry. (FMH is fetal red blood cell [RBC] volume; fetal blood volume is double [expected fetal hematocrit is 50%].) RESULTS: The fetal RBC volume diagnosed in maternal circulation after delivery ranged from insignificant FMH of not more than 0.1 mL to excessive FMH of 65.9 mL (median, 0.7; mean, 0.78; SD, 1.48). FMH of more than 2.5 mL (immunoglobulin [Ig] G anti‐D insufficient dose 50 µg) was observed in 1.4% (49/3457) and excessive volumes of FMH of more than 5 mL (insufficient dose, 100 µg) in 0.29% (10/3457). Delivery by cesarean section presented a higher risk of incidence of FMH of more than 2.5 mL (odds ratio, 2.2; p = 0.004) when compared with normal vaginal delivery. It did not, however, present a significant risk factor for the incidence of excessive volumes of FMH of more than 5 mL. CONCLUSION: During normal vaginal delivery as well as during delivery by cesarean section, FMH of less than 5 mL occurs in the great majority of cases, and thus for the prevention of D alloimmunization, an IgG anti‐D dose of 100 µg should be sufficient. Contrarily, only rarely does greater FMH occur and delivery by cesarean section does not present a risk factor.     

Heterogeneity of F cells in β‐thalassemia

BACKGROUND: Fetal hemoglobin (HbF), which is largely replaced after birth by the adult Hb, is concentrated in a few “F cells.” Their number significantly increases in certain physiologic and clinical situations, including in β‐thalassemia (β‐thal). Their quantification is used to detect fetal–maternal hemorrhage (FMH), where fetal cells enter the maternal circulation. We were confronted with a pregnant woman with β‐thal who was suspected to have FMH. To establish the usefulness of a flow cytometric procedure to differentiate between fetal cells and the maternal F cells, we screened adult β‐thal patients. STUDY DESIGN AND METHODS: Blood samples were simultaneously stained with fluorescent antibodies to HbF and to carbonic anhydrase (CA) isotype II, which is specific to adult red blood cells (RBCs). RESULTS: A heterogeneous distribution of RBCs with respect to HbF and CA expression was observed: adult non‐F cells (CA+HbF–) and F cells (CA+HbF+/HbF++) as well as F cells with characteristics of fetal cells (CA–HbF++). CONCLUSIONS: The presence of CA–HbF++ RBCs in nonpregnant women, and even men, with thal indicates that the CA/HbF method is inappropriate for detection of FMH. The coexistence of F cells carrying fetal or adult markers suggests that they originate from two types of stem cell, adult and fetal, lineages. Normally, the fetal lineage is insignificant, but in β‐thal, as HbF‐containing RBCs have a selective advantage, the “fetal” lineage gains significance.     

Fetal hemolytic anemia and intrauterine death caused by anti-M immunization

BACKGROUND: Antibodies with anti-M specificity are detected in 10 percent of pregnant women with a positive antibody screen, but anti-M is only rarely associated with hemolytic anemia in the fetus. STUDY DESIGN AND METHODS: This study reports on three pregnancies in one family that all resulted in severe fetal anemia. The first fetus died in utero with hydrops fetalis during the 20th gestational week and the second child was delivered after 28 weeks of gestation with hydrops fetalis and a hemoglobin level of 16 g per L whereas the third affected child was treated with intrauterine red cell (RBC) transfusions before delivery at 28 weeks of gestation. RESULTS: The direct antiglobulin test was negative but anti-M in a low titer was detected through the three pregnancies, and its clinical relevance, which initially was uncertain, was confirmed by pronounced in vivo hemolysis in maternal blood of chromate ((51)Cr)-labeled M+ RBCs and normal survival of (51)Cr labeled M- RBCs. CONCLUSION: It is concluded that anti-M immunization in a few cases may cause severe fetal hemolytic anemia and intrauterine death. It remains to be elucidated why a normally clinically insignificant antibody is this aggressive in a small proportion of cases. Because the condition is treatable, anti-M must be considered as a possible cause of fetal anemia and intrauterine death.     

Marked splenomegaly in fetal cytomegalovirus infection: detection supported by three-dimensional power Doppler ultrasound.

An enlarged fetal spleen can be associated with fetal infection, anemia and different syndromes but its prenatal diagnosis is rare. We report on a diagnosis of splenomegaly at 32 weeks' gestation in a fetus which was found to be affected by cytomegalovirus infection. An enlarged spleen was suspected when the stomach was found to be displaced anteriorly and medially and the diagnosis was supported on visualization of the splenic vessels by color and three-dimensional power Doppler ultrasound. The patient had been referred because of fetal growth restriction and intracerebral anomalies and the additional finding of splenomegaly was highly suspicious for cytomegalovirus infection. This was confirmed by positive maternal serology and by neonatal virus excretion in urine. Retrospectively, examination of stored blood samples from 9 and 23 weeks' gestation revealed an early cytomegalovirus infection. Antenatal and neonatal magnetic resonance imaging examinations showed microcephaly, lissencephaly and the presence of microcalcifications. At the age of 9 months, the child suffers from severe neurological impairment and blindness due to severe optical atrophy. This case emphasizes that color Doppler and three-dimensional power Doppler ultrasound can facilitate the antenatal diagnosis of splenomegaly and can help to delineate the spleen from the similar-looking neighboring liver.     

Detection of fetal hemorrhage in Rh immune globulin candidates. A rosetting technique using enzyme-treated Rh2Rh2 indicator erythrocytes

Current serologic tests occasionally fail to identify women needing more than one vial of Rh immune globulin. We compared the indirect antiglobulin test after incubation with anti-D and a rosetting technique using enzyme treated Rh2Rh2 erythrocytes as methods for identifying significant fetal maternal hemorrhage (FMH). Artificial mixtures containing 0.05 to 1.2 percent Rh1rh (CcDe) fetal red blood cells mixed with rh (ce) adult red blood cells were tested. The indirect antiglobulin test of the 0.6 percent mixture (approximately 30 ml FMH) was reported to be microscopically positive by 17/20 technologists; whereas, 20/20 found the rosetting test to be strongly positive. The volume of FMH in 118 postpartum Rh immune globulin candidates was quantified using Kleihauer's test and formula. The results of the rosetting and Kleihauer tests of blood specimens from these patients were negative 1.4 ml for two, and strongly positive rosetting test and FMH of 6.5 ml for one. The rosetting test utilizes routine blood banking skills and requires 5 minutes more "hands on" time than an indirect antiglobulin test. Confirmation and quantification of positive results by an acid-elution test is necessary.     

Middle Cerebral Artery Peak Systolic Velocity

OBJECTIVE: The aim of this work was to review the use of middle cerebral artery peak systolic velocity (MCA PSV) for the diagnosis of fetal anemia. METHODS: With the use of a computerized database (MEDLINE), articles on the diagnosis of fetal anemia with ultrasonography were reviewed. Other pertinent references were obtained from the references cited in these articles. In addition, my own institution's clinical experience of the past 18 years was reviewed. RESULTS: Several ultrasonographic parameters have been used to diagnose noninvasive fetal anemia. On the basis of robust data, the MCA PSV is the best ultrasonographic parameter used in the management of fetuses at risk for anemia due to different causes. It is also superior to amniocentesis for the diagnosis of fetal anemia in cases of red cell alloimmunization. CONCLUSIONS: Middle cerebral artery peak systolic velocity is effective for diagnosis of noninvasive moderate and severe fetal anemia. This parameter should not yet be considered the global standard of care for diagnosis of fetal anemia because incorrect use by an inexperienced operator may cause more harm than good; however, if there is a reasonably close medical center with sonographers or sonologists trained to assess the MCA PSV, patients at risk for fetal anemia should be referred to this center.     

Quantification of feto-maternal haemorrhage (FMH) by flow cytometry: anti-fetal haemoglobin labelling potentially underestimates massive FMH in comparison to labelling with anti-D

Many centres now routinely use flow cytometry to quantify feto-maternal haemorrhage (FMH). However, which flow cytometric method is the most accurate in quantifying FMH is currently unknown. An audit of clinical results in which FMH had been estimated by both directly conjugated monoclonal anti-D and anti-fetal haemoglobin (HbF) labelling suggested that the anti-HbF labelling method may underestimate massive FMH in comparison to labelling with anti-D. Subsequent to this audit, 46 samples of adult D-negative blood were spiked with varying amounts of D-positive cord blood (0.05-10% fetal cells per sample), and the number of fetal cells present was quantified by both labelling methods. The percentage of fetal cells detected by anti-D was not significantly different to the estimated percentage of fetal cells added to each sample (P = 0.636). However, anti-HbF labelling significantly underestimated the percentage of fetal cells present (P = 0.0001). In comparison to anti-D, the percentage of fetal cells detected by anti-HbF was also significantly lower (P < 0.0001). The difference in fetal cell detection between anti-D and anti-HbF labelling was only apparent in the spiked samples containing > or =1% fetal cells per sample. In samples containing < or =0.6% fetal cells, no significant difference in the detection of fetal cells between anti-D and anti-HbF labelling was observed (P = 0.11). To allow adequate immunoprophylaxis in D-negative mothers with massive FMH, we recommend that anti-D labelling should be used in the routine flow cytometric estimation of FMH.     

Use of maternal plasma for non-invasive prenatal diagnosis of fetal ABO genotypes.

BACKGROUND: Measurement of free fetal DNA in maternal plasma opened a door for non-invasive prenatal diagnosis. Prenatal diagnosis of fetal ABO genotypes can provide a basis for the prevention and therapy of maternal-fetal incompatibility. We identified fetal ABO genotypes using fetal DNA in plasma from pregnant women with blood group O. The aim of the study was to investigate the accuracy and feasibility of this method. METHODS: A total of 105 blood group O women in middle or late pregnancy were enrolled. Fetal DNA in maternal plasma and genomic DNA in umbilical vein blood from newborns were extracted using a QIAamp DNA Blood Kit. DNA was amplified to identify ABO genotypes by PCR with sequence-specific primers (PCR-SSP). The genotype results were evaluated using serologic tests for ABO phenotyping. RESULTS: Using DNA from umbilical vein blood, ABO genotypes of 105 newborns were successfully identified by PCR-SSP. Using fetal DNA from maternal plasma, 88.6% (93/105) fetal ABO genotypes was correct; 12 false results were from 66 pregnant women with fetuses of type non-O. The accuracy in middle pregnancy was lower than that in late pregnancy, although the difference was not significant (0.05相似文献   

Treatment of D alloimmunization in pregnancy with plasmapheresis and intravenous immune globulin: case report

The prevalence of D alloimmunization occurs between 0.15% and 0.4%. The anti-D can cross the placenta and cause hemolysis and fetal anemia. At present, a Doppler study of the middle cerebral artery allows the monitoring of the degree of fetal anemia. The treatment in cases of moderate to severe anemia in fetuses of less than 34–35 weeks of gestation is intrauterine transfusion via cordocentesis. However, with high titers of anti-D, in the absence of fetal anemia it is possible to modulate the maternal immune response by plasmapheresis and intravenous immunoglobulin administration. We present a case report of an Rh(D) alloimmunized pregnancy treated with plasmapheresis followed by intravenous immunoglobulin administration. We performed a caesarean section at 31 weeks, 5 days of gestation. The hemoglobin at birth was 13.8 g/dl and hematocrit 40.8%. Intrauterine transfusion was not necessary.     

Quantification of fetomaternal hemorrhage by fluorescence microscopy is equivalent to flow cytometry

BACKGROUND: The quantification of fetal cells in the maternal circulation remains an important goal to determine the amount of anti-D necessary to prevent active immunization of a D- mother giving birth to a D+ baby. Underestimation of fetomaternal hemorrhage (FMH) results in inefficient anti-D prophylaxis and maternal immunization; overestimation of FMH results in higher doses of passively transferred anti-D, higher costs, and the risk of disease transmission. Thus, a reliable method to quantitatively assess FMH is necessary. STUDY DESIGN AND METHODS: Serial dilutions of artificial FMH were quantitatively measured by three different methods: flow cytometry, fluorescence microscopy (each after anti-D staining), and by the Kleihauer-Betke test. The accuracy and precision of the three methods were compared by statistical analysis. RESULTS: Fluorescence microscopy and flow cytometry were comparably accurate and precise in quantifying FMH. In contrast, the accuracy of the Kleihauer-Betke test was poor, resulting in substantial overestimation of FMH in the samples with lower fetal cell concentrations. CONCLUSION: Anti-D flow cytometry and fluorescence microscopy for detection of fetal cells offer equally reliable and precise methods in contrast to the Kleihauer-Betke test. Fluorescence microscopy may be established as standard to quantify FMH in clinical practice because it is comparable to flow cytometry; in addition, it is time saving and is less expensive.     

P14Hereditary Persistence of Fetal Haemoglobin Leading to Inappropriate Anti‐D Prophylaxis at Delivery

Fetal maternal haemorrhage (FMH) during pregnancy or delivery can lead to maternal RhD immunisation. Using the Kleihauer‐Betke test, which detects fetal Hb (HbF) in cells, the necessary dose of prophylactic anti‐D can be given to prevent immunisation. Hereditary persistence of fetal haemoglobin (HPFH) is a benign condition where HbF production continues into adult life. Classification is pancellular when HbF levels are elevated in all red cells or heterocellular when HbF is confined to a sub‐population of cells. This is a rare condition, but these HbF cells may be mistaken for fetal cells in the Kleihauer test; we report such a case. Post‐partum samples from an RhD negative woman and her RhD positive infant were tested the day following delivery. 500 iu of prophylactic anti‐D was issued and a Kleihauer test performed to assess FMH volume and additional anti‐D requirement. The FMH was estimated as 33 mL, however the HbFcells appeared as ‘intermediates’ or partially eluted, raising suspicion of HPFH. A total of 4500 iu of prophylactic anti‐D was issued, since although HPFH was suspected, fetal origin of the HbF containing cells could not be excluded due to weekend unavailability of confirmatory testing. Subsequent flow cytometric (FACS) testing for FMH and haemoglobinopathy testing showed the patient to have previously undiagnosed HPFH. The timing of receipt and testing of these samples prevented confirmation of HPFH within the 72‐h deadline for anti‐D maximum effectiveness, leading to administration of precautionary additional anti‐D doses. The likelihood of HPFH was considered because of the appearance of the HbF cells as ‘intermediates’, however not all laboratory staff will be familiar with such cells, particularly if they have not had experience in a haemoglobinopathy laboratory. This possibility should be highlighted.