Amniotic Fluid Bilirubin Concentration Research Articles

Middle cerebral artery Doppler.

In the last 50 years we have seen tremendous advances in the management of fetal haemolytic anaemia. What was once a disease of high perinatal mortality and morbidity has become a condition readily amenable to antenatal diagnosis and intrauterine fetal therapy. Prior to the 1960s, the management of severe fetal anaemia relied on preterm delivery and subsequent therapy in the neonatal period. In 1961, Liley 1 described the use of amniocentesis to predict the severity of fetal haemolytic anaemia. Amniotic fluid bilirubin concentrations, reflecting the degree of fetal haemolysis, gave indirect assessment of fetal anaemia. The first therapeutic intervention for fetal anaemia was again described by Liley in 1963 2 . He performed the first intrauterine transfusion via the intraperitoneal route. The introduction of real‐time ultrasound facilitated needle‐guided intraperitoneal transfusion in 1977 3 . The first intravascular transfusion, performed fetoscopically, was carried out by Rodeck in 1981 4 . Developments in imaging technology mean that intravascular transfusion is now routinely performed with the aid of ultrasound guidance, and success rates of 92–94% can be expected for the non‐hydropic anaemic fetus 5 , 6 . In the last twenty years we have witnessed another milestone in the management of fetal haemolytic disease. Prediction of fetal anaemia by the non‐invasive method of middle cerebral artery Doppler has revolutionised the assessment of rhesus sensitised pregnancies, and provides the focus of discussion for this article. Rationale for middle cerebral artery Doppler studies in fetal anaemia The physiological response of a fetus to anaemia is an increase in stroke volume and hence an increase in cardiac output. As a result, the fetus develops a hyperdynamic circulation. Blood flow is redistributed to vital end organs, including brain, heart and adrenals, following changes in vascular resistance and a reduction in blood viscosity. The utility of Doppler studies in the assessment of fetal anaemia relies on the premise that provided the cross‐sectional area of a blood vessel remains constant, blood velocity is directly proportional to blood flow. In addition, decreased blood viscosity results in increased blood flow 7 , 8 . The increase in cardiac output, and reduced blood viscosity will both lead to in an increase in blood flow, and hence blood velocity, in the anaemic fetus. In the middle cerebral artery this is reflected by an increase in peak systolic velocity. The middle cerebral artery is the vessel of choice for assessment because it is accessible, and sensitive to the effects of hypoxia. The technique for middle cerebral artery peak systolic velocity measurement has been described in detail by Mari, et al. 9 , 10 . An axial section through the fetal head that includes the thalami and cavum septum pellucidum is obtained and the circle of Willis visualised with colour or power Doppler. The middle cerebral artery is examined close to its origin from the internal carotid artery. The sample volume should be placed in the centre of the vessel and the angle between the ultrasound beam and the direction of blood flow kept as close to 00 as possible. Angle correction can be used if required. The highest peak systolic waveform is measured, in the absence of fetal breathing movements. Open in a separate window Fig. 1 Insonation of middle cerebral artery, with angle correction. Measurement of peak systolic velocity (cm/s).

Bilirubin/Albumin Ratios in Fetal Blood and in Amniotic Fluid in Rhesus Immunization

To test the hypothesis that unconjugated bilirubin is equally distributed over the albumin molecules present in fetal blood and amniotic fluid in Rhesus (Rh) immunization. Molar concentrations of unconjugated bilirubin and albumin were measured in fetal blood and amniotic fluid samples, obtained before the first intrauterine transfusion in 30 nonhydropic, anti-D-alloimmunized fetuses, with gestational ages ranging from 20 to 35 weeks. Bilirubin concentration in amniotic fluid was best predicted by a combination of bilirubin concentration in fetal blood (P<.001), albumin concentration in fetal blood (P=.008), and albumin concentration in amniotic fluid (P<.001) (adjusted R2=0.91). The bilirubin/albumin ratios in fetal blood were linearly correlated with the bilirubin/albumin ratios in amniotic fluid (R2=0.75, P<.001). However, the bilirubin/albumin ratios in fetal blood were always higher than the bilirubin/albumin ratios in amniotic fluid (regression coefficient 1.4, 95% confidence interval 1.1-1.7). In our population, a bilirubin/albumin ratio in amniotic fluid of 0.10 or greater had a better sensitivity and specificity to predict severe anemia (Z hemoglobin -5 standard deviations or less) than the Queenan 4 or the Liley 2c line. The relation between fetal hemolysis and amniotic fluid bilirubin concentration is based on the linear correlation between bilirubin/albumin ratios in fetal blood and in amniotic fluid. The slope in Queenan's and Liley's chart follows that of the albumin concentration in amniotic fluid during gestation. III.

Effect of blood contamination on delta 450 bilirubin measurement: an in vitro comparison of two corrective methods.

Amniotic fluid bilirubin peaks (1.6–1.8 mg/L) at ∼19–22 weeks of gestation (1). An increase in amniotic fluid bilirubin concentration after 25 weeks indicates extensive hemolysis of fetal blood and potential erythroblastosis fetalis in which amniotic fluid bilirubin can reach ∼10 mg/L (2)(3). Bilirubin in amniotic fluid can be measured by direct spectrophotometry, most commonly by the “delta 450 bilirubin” method (4). Contaminating pigments, such as hemoglobin, can interfere in the method. Chloroform extraction has been used to extract bilirubin, leaving water-soluble pigments in the aqueous layer. The delta 450 bilirubin is then measured in the chloroform fraction and interpreted by use of the Liley plot (5)(6). A delta 410 correction formula also exists that theoretically accounts for hemoglobin interference. The delta 410 absorbance is measured and used to correct the delta 450 bilirubin (4)(6)(7). Our objective was to examine the effect of hemolyzed-whole-blood contamination on the delta 450 bilirubin measurement and to compare corrective methods. After receiving Institutional Review Board approval, we pooled amniotic fluid specimens collected for physician-ordered fetal lung maturity assessment (stored at −20 °C). Visibly bloody or meconium-contaminated samples were not used. The pool was centrifuged at 1100 g for 5 min, and the supernatant was filtered through a 22 μm AP 20 prefilter (Millipore Corp.) and a 1.2 μm RA filter (Millipore). Bilirubin was undetectable in the pool by delta 450 measurement. We prepared a 600 mg/L stock solution of unconjugated bilirubin (ICN Biomedicals Inc.) by dissolving 6.0 mg of bilirubin in 0.1 mL of dimethyl sulfoxide (Sigma Chemical Co.) and 0.2 mL of sodium carbonate (100 mmol/L). The solution was vortex-mixed until the bilirubin was dissolved. We then added 9.5 mL of pooled amniotic fluid, followed by 0.2 mL of HCl (100 mmol/L) to …

On the Origin of Amniotic Fluid Bilirubin

We studied the relationship between bilirubin concentrations in amniotic fluid and fetal blood in 68 non-hydropic rhesus d-alloimmunized anemic fetuses at first blood sampling. In these alloimmunized fetuses, the amniotic fluid/fetal blood ratio for bilirubin decreased from 0.09 at 28 weeks to 0.05 at 33 weeks. In normal fetuses, amniotic fluid/fetal blood ratios for bilirubin, and for albumin, are in the same range and show a similar decrease during gestation. We conclude that amniotic fluid bilirubin concentration is determined, firstly, by fetal blood bilirubin concentration and, secondly, by the amniotic fluid/fetal blood ratio of albumin. Among five possible pathways bilirubin could take to build up a concentration in amniotic fluid (fetal kidneys, lungs, skin, bowel, membranes), the intramembranous pathway is the only one that is compatible with the amniotic fluid/fetal blood ratios for bilirubin that we found and must therefore be the most important.

Bilirubin in amniotic fluid does not interfere with the Abbott TDx FLM II assay.

Bilirubin, a breakdown product of lysed red blood cells, is present in amniotic fluid in very small concentrations relative to serum (1). In an uncomplicated pregnancy, bilirubin in amniotic fluid peaks at ∼19–22 weeks of gestation at concentrations of 1.6–1.8 mg/L (1). Rh isoimmunization and its severe manifestation, erythroblastosis fetalis, are associated with intrauterine hemolysis, which leads to increases in amniotic fluid bilirubin concentrations (2) up to 9.6 mg/L (3)(4). The assessment of fetal lung maturity (FLM) in these pregnancies is sometimes necessary, but the ability to utilize current methods in the presence of increased bilirubin concentrations is unclear. The commercial TDx FLM II assay (Abbott Laboratories), which is based on fluorescence polarization technology and the dye PC-16 (5), is the most commonly used method to assess FLM (6). Although the manufacturer does not recommend testing icteric amniotic fluid samples, nothing has been published about the nature or quantification of their interference with the TDx FLM II assay. Our objective was to examine the effect of bilirubin on TDx FLM II concentrations. Frozen leftover amniotic fluid samples sent to the laboratory for physician-ordered FLM testing …

Iterative, Spectrophotometric Method for Determination of Amniotic Fluid Bilirubin Concentrations: Comparison with the Liley Method

Since 1961, the Δ A 450, or Liley, method has been used to estimate amniotic fluid (AF) bilirubin concentrations in RhD-alloimmunized pregnancies (1). Although the results of this method often predict the degree of fetal anemia relatively well, blood-contaminated AF may give incorrect results (2)(3) Furthermore, with this method the AF bilirubin content is not expressed quantitatively as the bilirubin concentration. As an alternative, a diazotization method has been used to measure the bilirubin concentration in AF (4). Blood contamination could produce falsely increased bilirubin concentrations with this diazotization method, however, and this method is also considered to be inaccurate at the very low bilirubin concentrations in AF (3). Recently we developed a procedure that uses a mathematic step-by-step (iterative) procedure to deconvolute the superimposed absorbance scans of pigments in cerebrospinal fluid into the scans of individual pigments. This iterative model is now in routine use for the spectrophotometric determination of methemoglobin (metHb), oxyhemoglobin (oxyHb), and bilirubin concentrations in cerebrospinal fluid (5). The method is fast and simple, corrects for the relative interference of each of these components on the others, and has a lower limit of detection of 0.1 μmol/L for each of these components. We suggest that, because of its high sensitivity, this method may become a reliable procedure to measure bilirubin concentrations in AFs. AF was obtained after informed consent from patients [n = 52; mean (SD) gestational age, 30.8 ± 3.6 weeks] whose pregnancies were complicated by red cell alloimmunization. The fluids were centrifuged at 1400 g for 10 min and were protected from light before analysis. Measurements were performed with a UltrospecPlus spectrophotometer (Amersham Pharmacia Biotech). For the Liley method, absorbance was measured at 365, 450, and 550 nm, and a baseline was estimated between 365 and 550 nm on a …

Noninvasive assessment of fetal anaemia

In pregnancies complicated by severe red cell alloimmunization, fetal haemolytic anaemia can lead to intrauterine demise as early as 17 weeks' gestation. Intrauterine intraperitoneal blood transfusion, introduced by Liley in 1963, proved to be the first successful example of fetal therapy. The use of this complex procedure, involving X-ray guided puncture of the fetal peritoneal cavity, was limited to fetuses with a gestational age ranging from 24 to 33 weeks. When performed below 26 weeks, survival rates were as low as 16%. The need for intrauterine transfusion was mainly based on determination of bilirubin levels in amniotic fluid, and plotting the results in a three-zone chart devised by Liley. This chart was based on correlations of amniotic fluid bilirubin concentrations with the degree of neonatal anaemia, and ranged from 27 to 41 weeks' gestation. In the 1980s, with the introduction of ultrasound-guided intravascular transfusion, treatment became possible from 17–18 weeks' gestation onwards. Consequently, there was a need for diagnostic tests to assess the degree of anaemia in the second trimester fetus. The use of the Liley chart was shown to be unreliable in the prediction of disease severity before 27 weeks' gestation. Ultrasonographic detection of fetal hydrops in these pregnancies reliably indicates severe anaemia, but for reasons that are still unclear, a significant number of severely anaemic fetuses do not show ultrasonographic signs of hydrops. In 1988, Nicolaides et al studied the use of several ultrasonographic parameters to predict anaemia in nonhydropic fetuses. Their results were disappointing, and they concluded that the only reliable diagnostic test to assess the degree of fetal disease was fetal blood sampling.

Erythropoietic suppression in fetal anemia because of Kell alloimmunization

Objective: Our purpose was to test the hypothesis that maternal anti-Kell alloimmunization produces fetal anemia by erythrold suppression.Study Design: ErYthropoiesis in 11 anemic fetuses from maternal anti-Kell alloimmunization was compared with that in 11 fetuses where the mother was alloimmunized to RhO; each was matched for hematocrit, gestational age, hydrops, and perinatal outcome. Comparisons of the difference were performed by either paired t or Wilcoxon tests.Results: The anti-Kell group had reduced reticulocytosis (p = 0.007) and erythroblastosis (p = 0.045) and lower amniotic fluid bilirubin concentrations (p = 0.02) in comparison with the anti-O group. No correlation was found between hematocrit and reticulocytosis in the anti-Kell group, whereas the anti-O group had a significant-linear relationship (r = 0.63, p < 0.05), indicating a progressive reticulocytosis in response to the degree of anemia.Conclusion: These findings suggest that erythroid suppression, rather than hemolysis, is the predominant mechanism in producing fetal anemia related to maternal Kell alloimmunization. Fetal blood sampling is the investigation of choice in the evaluation of anemia related to maternal Kell alloimmunization, because reduced hemolysis means amniotic fluid bilirubin concentrations correlate poorly with anemnia.

8 Alloimmune conditions and pregnancy

Transfer of fetal red blood cells and platelets to the maternal circulation can stimulate an immune response with production of immunoglobulin that can cross the placenta. Similarly, passage of maternal stem cells to an immunologically incompetent fetus can theoretically produce graft-versus-host disease. disease. Maternal sensitization to red blood cell antigens such as D and Kell can result in anaemia, hydrops, and death in an incompatible fetus. Current assessment of these pregnancies involves serial analysis of amniotic fluid bilirubin concentration, with umbilical cord blood sampling reserved for special circumstances; neither ultrasound or Doppler blood flow analysis are accurate in the prediction of fetal haematocrit. Intravascular transfusion is the treatment of choice for hydropic fetuses. Perinatal survival in non-hydropic fetuses is similar with either intravascular or intraperitoneal transfusion, and the choice of procedures is individualized. Isoimmune fetal thrombocytopenia is usually the result of maternal sensitization to the PlA1 antigen. There is significant risk of intracranial haemorrhage, both antepartum and during labour and delivery. Umbilical cord blood sampling at term can determine fetal platelet count and the need for platelet transfusion, and can aid in deciding the appropriate route of delivery.

Relation of the site of congenital upper gastrointestinal obstruction to amniotic fluid bilirubin concentration.

AbstractIn order to ascertain relative amniotic fluid bilirubin concentrations and to assess their value in antenatal diagnosis of upper gastrointestinal obstruction, amniotic fluid samples obtained from eight women who gave birth to infants with congenital upper gastrointestinal obstruction were analyzed spectrophotometrically.Types of congenital upper gastrointestinal obstruction in the study included esophageal atresia (1), pyloric atresia (1), high jejunal atresia (1), annular pancreas (2), and duodenal atresia (3).Amniotic fluid bilirubin concentration of the six women who gave birth to infants with intestinal obstruction distal to Vater's ampulla increased above the mean + 2 S.D. of the control. By contrast, the two who gave birth to infants with esophageal atresia or pyloric atresia had normal levels of amniotic fluid bilirubin.These findings suggest that when the obstruction is distal to Vater's ampulla, bile is regurgitated into the amniotic fluid by the fetus, resulting in a significant increase in amniotic bilirubin concentration. However, amniotic fluid bilirubin doesn't originate from the upper gastrointestinal tract in normal pregnancies: its concentration was within the normal range in the two fetuses with esophageal or pyloric atresia who couldn't regurgitate bile into the amniotic fluid.

Ultrasound-guided Fetal Intravenous Transfusion for Severe Rhesus Haemolytic Disease

Intraperitoneal transfusion of unborn infants with severe rhesus hemolytic disease gives poor results when the fetus is of low gestational age and has hydrops. Direct intravascular transfusion of fetuses under feto-scopic control at 23 to 26 weeks of gestation is more efficient, but it cannot be used beyond that age because of fetoscopic limitations. The authors have therefore tried direct intravenous transfusion of fetuses through the hepatic part of the umbilical vein using a thin needle guided by ultrasound. The procedures conformed with the Declaration of Helsinki of 1975. Results in two cases are described. Case 1, a 33-year-old woman (blood group O, rhesus-negative) was in her fifth pregnancy. Ultrasonic assessment showed a fetus of normal size for 29 weeks. Fetal ascites was present, and the amniotic fluid bilirubin concentration was 14 μmol/liter(0.8 mg/100 ml), which was in the “severely affected” zone of the prognostic chart. The patient was treated with betamethazone (12 mg daily for 2 days), ritodrine (10 mg four times daily), and phe-nobarbitone (100 mg daily). An intraperitoneal transfusion was planned, with 50 ml of group O, rhesus-negative (cde/cde) erythrocytes suspended in sterile isotonic sodium chloride (packed cell volume, 0.60 (60 per cent)). To prevent immunological complications, the cell suspension was irradiated with 2160 rad for 4 minutes before use. During the transfusion, the authors observed a dilated umbilical vein and gave a further 10 ml of blood into the hepatic part of the vein. A fetal blood sample, which looked like reddish serum, showed the fetus to be group A, rhesus-positive, and gave a strongly positive reaction to a direct Coombs test. A second intravenous transfusion of 25 ml was given a week later. A fetal blood sample before the second transfusion showed a ratio of fetal to adult erythrocytes of 37:63. At 32 weeks, a girl of 1 700 g was delivered by cesar-ean section. The Apgar score was 3 at 1 minute and 10 at 5 minutes. The umbilical cord hemoglobin concentration was 6.4 g/dl and the bilirubin concentration 144 μmol/liter (8.4 mg/100 ml). The ratio of fetal to adult erythrocytes was 22:78. The infant had a slight bluish discoloration around the umbilicus, possibly a slight he-matoma. During the next 2 days, five exchange transfusions were given because of high and rising serum bilirubin concentrations, the highest being 393 μmol/liter (22.9 mg/100 ml). There were no further complications. Case 2, a 25-year-old woman in the 23rd week of pregnancy, was admitted for termination of pregnancy after a prenatal diagnosis of genetic disease (chromosomal abnormality). There was no evidence of rhesus isoimmunization. Ultrasound-guided puncture for fetal blood sampling was performed. Biparietal diameter was compatible with the age of gestation. Puncture of the hepatic part of the umbilical vein was performed under epidural anesthesia. The tip of the needle was visualized on the oscilloscope in the lumen of the vein and 0.5 ml of blood was withdrawn. Analysis on a Coulter counter showed 100 per cent fetal blood. Subsequently, 40 mg of dinoprost trometamol (Amoglandin) were instilled into the amniotic cavity for termination. Knowledge of various factors—for example, the size of transfusion, the cardiovascular effects of transfusion, the importance of different blood variables in fetal blood—should be established, but it is hoped that direct intravenous fetal tranfusion will improve the prognosis of high risk, rhesus-sensitized fetuses that cannot be saved by traditional methods.

A CRITICAL REVIEW OF INTRAUTERINE FETAL TRANSFUSION

A critical review of 142 intrauterine fetal transfusions performed in 99 patients during 107 pregnancies raised doubts about the absolute benefit of the procedure. Two main problems emerged: the diagnostic criteria upon which decisions were based were inadequate and the fetal mortality associated with transfusion was high, especially when this was performed before 28 weeks. We measured amniotic fluid bilirubin concentration by a biochemical method but the results following intrauterine fetal transfusion were similar to those observed by others using spectrophotometric examination of amniotic fluid.