Amniotic Fluid Bilirubin 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).

Rhesus Alloimmunisation: Forgotten but not Gone

The prophylactic use of rhesus immunoglobulin has led to a significant reduction in the incidence of haemolytic disease of the newborn secondary to rhesus alloimmunisation, which now only occurs in 1 in 1000 live births. Despite prophylaxis, antibodies to the D antigen are still the commonest cause of rhesus isoimmunisation. Although now a rare pregnancy disorder rhesus isoimmunisation continues to be a challenging obstetric complication and while the face of this disease has undergone many changes over recent years, the principles of management remain unchanged. Pregnancies at risk of the disease are identified by past obstetric history and the antenatal detection of red cell antibodies with serial measurement of antibody levels. In cases of heterozygous paternity the real risk of fetal disease can be determined using new DNA techniques to determine the fetal blood cell type with tissue obtained from amniocentesis, and now by molecular analysis of circulating fetal DNA in the maternal plasma and serum. Sensitised pregnancies are monitored for the development of fetal anaemia. Since its introduction into clinical practise by Liley over 40 years ago, spectral analysis of amniotic fluid bilirubin as an indirect measure of fetal haemolysis has been the primary method of screening for fetal anaemia. Noninvasive evaluation is now possible with the use of doppler ultrasound to detect changes to fetal blood flow secondary to fetal anaemia, most commonly using the middle cerebral artery, and ultrasound evaluation of fetal haemopoetic organs and for the diagnosis of hydrops. Ultrasound guided intrauterine transfusion of red blood cells remains the treatment for the severely affected fetus. Perinatal survival rates in the non hydropic fetus are reported at over 90% and long term studies have revealed normal neurological outcomes for more than 90% cases.

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.

Elevated Plasma Corticotrophin Release Factor Levels and In Utero Meconium Passage

Intrauterine meconium (MEC) passage and aspiration may result in significant newborn morbidity, though there is little understanding of the physiologic mechanisms for MEC passage. We hypothesized that stress induces fetal MEC passage via corticotrophin releasing factor (CRF), a known mediator of colonic motility in adult rats. Pregnant rats at e22 were subjected to acute hypoxia or normoxia for 35 min, after which rats were anesthetized and fetuses operatively delivered. Amniotic fluid bilirubin and intestinal alkaline phosphatase were measured as markers for MEC passage, and fetal and maternal plasma CRF and corticosterone levels determined. Hypoxic stress induced defecation in all dams and provoked visible MEC passage in all fetuses. Amniotic fluid bilirubin content was significantly higher in hypoxic fetuses versus controls (1.064 +/- 0.101 versus 0.103 +/- 0.003 O.D. at 410 nm) and intestinal alkaline phosphatase was consistently elevated in MEC stained amniotic fluid. Hypoxia significantly increased plasma CRF (maternal, 82 +/- 5 to 196 +/- 14 pg/mL; fetal, 284 +/- 15 to 1523 +/- 185 pg/mL) and corticosterone (maternal, 417 +/- 50 to 1150 +/- 50 ng/mL; fetal, 96 +/- 5 to 182 +/- 10 ng/mL) compared with controls. In view of the known action of CRF in adult colonic motility, these results suggest that hypoxic stress-mediated MEC passage in term fetal rats is mediated by a CRF dependent pathway.

EL DOPPLER DE LA ARTERIA CEREBRAL MEDIA REEMPLAZA A LA ESPECTROFOTOMETRÍA DEL LÍQUIDO AMNIÓTICO COMO EL ESTÁNDAR EN EL MANEJO DE LA ISOINMUNIZACIÓN FETAL

SUMMARY Fetal anemia could be a devastating condition to the fetus. The main cause of fetal anemia is red-cell alloimmunization. The standard of care for this condition has been the serial amniocentesis to measure bilirubin levels by spectrophotometry of the amniotic fluid. Amniotic fluid bilirubin measurements are of limited value in the second trimester and in other causes of fetal anemia, besides amniocentesis is an invasive method with fetal risk. During the last 10 to 12 years a non invasive method to predict fetal anemia become available: measurement of the peak systolic velocity of the middle cerebral artery. Increased velocity may reflect fetal anemia. Here we present the evidence to support the use of this non invasive method to replace amniocentesis as the standard care in the management of red-cell alloimmunization and other causes of fetal anemia.

Evaluation of the Efficacy of Chloroform Extraction of Amniotic Fluid Bilirubin

The presence of increased concentrations of bilirubin in amniotic fluid is an indicator of fetal hemolysis (1)(2). In the Δ A 450 (or ΔOD450) method, the absorbance at 450 nm is used as a surrogate for concentration of amniotic bilirubin. Chloroform extraction of bilirubin from amniotic fluid samples can be used to eliminate spectral interference from hemoglobin, which absorbs at a maximum at 410 nm. However, clinical interpretation of the Δ A 450 often relies on studies in which chloroform extraction was not used (3)(4). We studied the extraction of bilirubin from 37 amniotic fluid samples submitted for bilirubin scanning. All studies were in accordance with the guidelines approved by the Institutional Review Board of the University of Utah. Frozen (−70 °C), light-protected samples were thawed and centrifuged at 900 g for 5 min at 2–6 °C. Absorbances of samples were scanned (Beckman DU-800) from 350 to 550 nm in 1-mL air-blanked quartz cuvettes. The use of water or chloroform blanks increased Δ A 450 <0.01 in both native and chloroform-extracted samples (n = 4). The Δ A 450 was determined as the difference between the absorbance at 450 nm and a logarithmic …

Determination of Amniotic Fluid Bilirubin Absorbance in Specimens Contaminated by Blood

The careful study of Foster et al. (1) reminds us of the difficulty of assessing amniotic fluid bilirubin absorbance in specimens contaminated by blood. However, it is not correct, as Foster et al. conclude, that bilirubin absorbance cannot be appropriately assessed in specimens contaminated in this manner. To eliminate interference from nonbilirubin pigments, my colleagues and I (2) exploited the fact that bilirubin is highly photolabile whereas interfering chromagens such as hemoglobin and methemoglobin are remarkably photostable. A few minutes of exposure to an intense light source (such as that from an ordinary slide projector) eliminates most of the bilirubin absorbance and allows the absorbance present before irradiation to be determined as described in the next paragraph. Slide projectors of a type similar to the one used here continue to be manufactured, e.g., the Kodak Carousel 4600 (Eastman Kodak Company) or the Vivitar 5000 AF (Vivitar USA). These and similar projectors …

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.

Rhesus disease and non-immune hydrops

Hippocrates first documented the condition of ‘fetal hydrops’ in 400 BC. There was recognition of jaundice of the newborn in the 17th century, but it was not until 1909 that Buchan established a link between some cases of newborn jaundice and anaemia, and 1932 before Diamond described the condition of erythroblastosis fetalis. Between 1938 and 1941, the identification of maternal red cell antibodies, the Rhesus antigen and blood group inheritance were established. In 1947, Diamond first described post-natal treatment utilizing transfusion, with exchange transfusion being described by Mollison in 1952. Intra-uterine therapy was not established until 1963 when Liley first published the use of intraperitoneal transfusion. The last 40 years have seen rapid advances in the understanding, assessment and therapy of Rhesus disease and non-immune hydrops. We have seen exploration of: amniotic fluid bilirubin levels, to determine whether a fetus is at risk of haemolytic disease of the newborn (Freda and Liley); plasmapheresis (Powell), aiming to reduce the maternal circulating antibody levels; fetal blood sampling, to establish a diagnosis; and intraperitoneal or intravascular transfusion, as methods of direct therapy, the latter initially using fetoscopy and then using an ultrasound-guided approach to fetal vessels. Stem-cell therapy has been explored and, more recently, non-invasive methods to assess fetal anaemia have been introduced using the mean range on a computerized cardiotocograph (CTG) and middle cerebral artery maximum flow velocity. In cases where the partner is heterozygous for a particular red cell antigen, the potential for determining fetal group by identification of fetal cells in the maternal circulation for testing has also become a possibility. Approximately 20% of cases of non-immune hydrops remain ‘idiopathic’, the remainder either being a result of a cardiac structural anomaly, arrhythmia or a metabolic disorder. Twinning accounts for approximately 6% of cases of non-immune hydrops. This article aims to give an overview of what is happening today and a glimpse of what might be possible in the future.

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 …

Invasive versus non-invasive testing in red-cell alloimmunized pregnancies

Amniocentesis for amniotic fluid bilirubin levels is the most widely used test to predict the severity of fetal disease in red-cell alloimmunization. Many textbooks and guidelines recommend serial amniocentesis to monitor these pregnancies. However, the reliability of amniotic fluid bilirubin measurements has been questioned. Two strategies have been proposed by investigators. Some advocate liberal or primary use of fetal blood sampling, while others promote the use of non-invasive ultrasonography and Doppler. The purpose of this literature review is to examine the usefulness of amniocentesis, ultrasonography and Doppler to predict the degree of fetal hemolytic anemia. The evidence suggests that amniotic fluid bilirubin measurements are of limited value in the second trimester. Furthermore, critical appraisal of the very few prospective studies is hampered by limitations in design or insufficient data given by the authors. Many ultrasound and Doppler parameters have been proposed as useful indicators of fetal anemia. The most promising of these methods are Doppler assessment of umbilical venous and middle cerebral artery flow velocities. We speculate that the accuracy of these non-invasive tests may be good enough to incorporate them in management protocols, possibly replacing amniocentesis. Well-designed prospective studies are needed to prove this hypothesis.

Assessment of the clinical usefulness of the ‘Queenan’ chart versus the ‘Liley’ chart in predicting severity of rhesus iso-immunization

In 1961 Liley developed a chart of changing amniotic fluid bilirubin levels (delta OD450) and gestation, with three zones delineating the severity of rhesus disease. This chart ranged from 27 to 40 weeks and was found to be clinically useful. Extrapolating the use of the Liley chart to earlier gestations however, was unsuccessful. Currently, cordocentesis is the only reliable means of assessing the fetal condition accurately prior to 27 weeks. In 1993, Queenan proposed a chart of delta OD450 from 14 to 40 weeks, with four zones to guide management. The aim of the current study is to assess the clinical usefulness of the 'Queenan' chart vs. the 'Liley' chart. There were 35 pregnancies affected by rhesus disease between 1990 and 1997 at the Mater Mothers' Hospital, Brisbane. The quantitative anti-D and delta OD450 levels obtained before intra-uterine transfusions were recorded. Each sample was labelled with the fetal condition at the time the sample was taken. Of the 72 delta OD450 samples, 36 (50 per cent) were performed before 27 weeks, and these included all four of the severely affected samples and 11 of the 13 moderately affected samples. The sensitivity of the Queenan chart in predicting the severely affected pregnancies was 100 per cent, with specificity of 79.4 per cent, positive predictive value of 22.2 per cent and negative predictive value of 100 per cent. For prediction of moderate/severely affected pregnancies, the sensitivity was 83.3 per cent, with specificity of 94.4 per cent, positive predictive value of 83.3 per cent and negative predictive value of 96.3 per cent. In conclusion, an delta OD450 chart which includes gestations less than 27 weeks is necessary with our changing caseload of rhesus iso-immunized patients, where severely affected pregnancies seemed to occur early and intervention even in moderately affected pregnancies seemed appropriate. We found that the Queenan chart is a suitable alternative to the Liley chart.

Hemolytic disease of the fetus: a comparison of the Queenan and extended Liley methods

Objective: To compare the clinical utility of the Liley and Queenan methods to monitor the severity of fetal hemolytic disease. Methods: Amniotic fluid bilirubin was measured in specimens from 73 women sensitized to red blood cell antigens. Chloroform-extracted amniotic fluid was evaluated spectrophotometrically for bilirubin content by using the change-from-expected value of the optical density at 450 nm. Values in the four Queenan zones were compared with those of the four zones of the Liley graph (middle zone subdivided). Clinical utility and accuracy of the two methods were compared. Results: Treatment was based on interpretation of bilirubin values plotted on the Liley graph. Hydrops fetalis was not observed. The highest value for each patient was significantly more likely to be plotted in the highest zone using the Queenan method (23 of 73 compared with eight of 73 patients; P < .001). Overestimation of risk occurred with greater frequency when using the Queenan method (13 of 67 compared with seven of 67 patients; P = .031). Overestimation of risk by the Queenan method also was more likely at or before 28 weeks’ gestation (10 of 49 compared with four of 49 patients; P = .031). In nine cases (13%), the Queenan graph and method would have prompted unnecessary or premature umbilical vein sampling that was withheld using the Liley graph. Conclusion: The performance of the linearly extended Liley graph was superior to that of the Queenan graph, because the Queenan method frequently overestimated risk.

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.

Analysis of Amniotic Fluid by UV Derivative Spectrophotometry

Abstract A direct method for evaluating bilirubin, albumin and oxyhemoglobin in amniotic fluid, is described. The procedure, which uses first and second UV derivative spectrophotometry, was carried out on a number of human specimens and laboratory mixtures. Special attention was given to bilirubin analysis when investigating the effects of photodegradation and of contaminating blood obtained by amniocentesis. The bilirubin results were also compared with those obtained by currently used colorimetric and extractive methods. The accuracy and precision of the method were estimated.

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.

Evaluation of standard parameters to predict exchange transfusions in the erythroblastotic newborn.

During the time period 1983-90, 91,300 consecutive pregnancies were monitored for red cell alloimmunization. Once revealed, the immunizations were followed by means of repeated maternal antibody titers, maternal anti-D quantitation in D-immunized women, amniotic fluid bilirubin levels and fetal hemoglobin concentrations. High dose intravenous immunoglobulin and/or intrauterine intravascular transfusion was given to prevent or treat fetal anemia. Delivery was induced for all before term when antibody titers were > or = 16. Nevertheless, exchange transfusions were performed in 41 newborns with mothers alloimmunized to Rh(D), Rh(c), Rh(E) and Kell antigens. Eight of the mothers were Rh(D) positive. Phototherapy alone was given to 35 newborns. Both maternal antibody titers and amniotic fluid bilirubin levels were found to be unreliable to predict the need of exchange transfusions in the newborns. Quantitation of maternal anti-D concentration was found to be significantly better predicting 62% at a cut-off level of 0.7 microgram/mL. Analysis of fetal hemoglobin concentration by cordocentesis is the only direct method to evaluate the degree of fetal affection, and should probably be performed when maternal antibody titers are > or = 64, anti-D concentration is > or = 0.7 microgram/mL and data indicate an aggravation of the immunization.

Severe Hemolytic Disease of the Newborn Caused by Anti-Gonzales Antibody

We report on a female infant with severe hemolytic disease of the newborn (HDN) secondary to anti-Gonzales antibody (anti-Go(a)) necessitating an exchange transfusion within the first day of life. The infant was born to a mother known to be Gonzales-antigen negative and a father who was Gonzales-antigen positive. The mother had an anti-Go(a) titer of 1:256 at 35 weeks' gestation. The infant was noted to have jaundice shortly after birth, with a bilirubin of 17.8 mg/dl (total) and .05 mg/dl (direct) at 11 hours of life. Coombs' test was positive and cord cells were Gonzales-antigen positive. Eluate on cord cells demonstrated anti-Go(a). Despite aggressive phototherapy, the total bilirubin reached 23.3 mg/dl by 24 hours of age and a double-volume exchange transfusion was performed. Following the exchange transfusion, phototherapy was continued for several days. The hemoglobin, which was 19.8 gm/dl at 11 hours of age, remained stable through the hospitalization, and no further transfusions were required. History revealed that two prior pregnancies resulted in Gonzales-antigen positive infants. The first child experienced mild jaundice requiring no therapy, and the second child required phototherapy for prolonged hyperbilirubinemia. Previous reported cases of anti-Go(a) suggest that this is not a cause of severe HDN. However, in view of the current case, it may be prudent to follow women with anti-Go(a) prenatally with amniotic fluid bilirubin studies, serial antibody titers, and fetal hemoglobin levels.