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Objective: To identify the types of requests, the patterns of testing, and the error rates associated with the genetic identification of fetuses at risk for immune-mediated hemolytic disease., Method: Retrospective review of the laboratory information system for all fetal blood group genotyping tests performed at Mount Sinai Hospital, Toronto, Canada from January 1997 to December 2006., Results: Amniotic fluid-derived DNA, from 220 women (243 pregnancies), was tested for one or more antigens (279 tests) when the father was heterozygous for the inferred blood group antigen or was unavailable. The PCR amplification failure rate of amniotic fluid-derived DNA was 5.0%. When the father was considered hemizygous for RHD or was not available, the fetus was positive in 68.6% and 72.7% of cases, respectively. Two amniotic fluid-derived DNA KEL1 SSP-PCR results did not correlate with the result determined from cultured amniocyte DNA., Conclusions: Paternal RHD hemizygosity may be overestimated. Thus, we recommend that RHD zygosity be established by molecular analysis of the RHD breakpoint. Cultured amniocytes should be reserved for testing if the amniotic fluid-derived DNA is inconclusive due to PCR amplification failure. We use PCR-RFLP genotyping methods for RHCE*E/RHCE*e (Rh E/e), KEL1/KEL2 (K/k), FYA/FYB (Fy(a/b)), and JKA/JKB (Jk(a/b)).

Objective: To assess the diagnostic accuracy of amniotic fluid Delta OD 450 values in the second and third trimesters of D-alloimmunized pregnancies., Methods: We searched our database for singleton D-alloimmunized pregnancies with nonhydropic fetuses, where amniocentesis was performed within 4 days of first fetal blood sampling. Amniotic fluid Delta OD 450 values were plotted on an extrapolated Liley's chart. Sensitivity and specificity were calculated for two commonly used cutoff levels, Liley's zone 3 and the upper third of Liley's zone 2. Severe fetal anemia was defined as a hemoglobin concentration of more than 5 standard deviations below the normal mean for corresponding gestational age., Results: Seventy-nine pregnancies met our inclusion criteria. Overall accuracy of the extrapolated Liley's curve in predicting severe fetal anemia was 75% (95% confidence interval [CI] 64, 84) for zone 3 and 86% (95% CI 77, 93) when the upper third of zone 2 was included. Sensitivity of Delta OD 450 values in Liley's zone 3 or the upper third of Liley's zone 2 was 95% (95% CI 74, 100) before and 98% (95% CI 89, 100) after 27 weeks., Conclusion: Liley's extrapolated curve predicts severe fetal anemia with reasonable accuracy and high sensitivity.

The aim of this work was to determine the presence of the RHD gene in fetal cells obtained from amniotic fluid (AF). We studied 65 samples of AF, 11 from RhD- mothers sensitized with anti-D. The fetal origin of the DNA was confirmed with the analysis of 1 VNTR locus and 3 STR loci in DNA samples from AF and maternal blood. The RHD genotyping was performed in non contaminated samples (n = 62) using a multiplex PCR strategy that yields 3 amplification products from RhD+ phenotypes and 1 DNA fragment from RhD- phenotypes. We genotyped 54 RhD+ fetuses (8 from RhD- sensitized mothers) and 8 RhD- fetuses (3 from RhD- sensitized mothers). Fetal DNA genotyping allows the diagnosis, from a single amniocentesis, of fetuses at real risk of hemolytic disease of the newborn. When the fetus is determined to be RhD- all invasive procedures can be avoided.

Relaxin was measured in serum and amniotic fluid of 136 pregnant women between the 12th and 38th gestational week by means of a new human relaxin-RIA. The pregnancies consisted of 111 pathology-free single fetuses, 10 with rhesus incompatibility, 7 with chromosomal aberration and 8 with sonographic diagnosed abnormalities. Relaxin could be detected in all samples tested the levels being ten times lower in amniotic fluid compared to serum. Serum relaxin levels showed a slight but not statistically significant decrease with increasing gestational age, in amniotic fluid relaxin values were consistent over the course of pregnancy. The ratio of amniotic fluid to serum relaxin displayed a statistically significant increase from the 12th to 23rd week of pregnancy. Individual courses of relaxin concentration in amniotic fluid revealed only low intra-individual variations but distinct inter-individual differences.

In Rhesus incompatibility, prenatal RhD typing of the fetus requires intrauterine blood sampling by cordocentesis or by chorionic villus biopsy. Amniocentesis is easier to perform, and carries a lower risk of enhancement of maternal immunization. Therefore, we evaluated polymerase chain reaction (PCR) for fetal RhD typing in amniocytes which were isolated from amniotic fluid (18-40 gw) obtained by amniocentesis (n=26) or during delivery (n=27). In the clinically most important group of children from RhD-negative women (n=25) and in 28 newborns of RhD- positive mothers, we found a 100 percent agreement between the findings of PCR and the results of serologic typing. If these encouraging results are confirmed in a larger series, the method could be used for the clinical management of RhD-negative women with Rhesus incompatibility and a heterozygous RhD-positive partner.

Objectives: Our purpose was to study deviations in values of amniotic fluid optical density at 450 nm in Rh-immunized pregnancies in the second and third trimesters and to propose a clinical management plan., Study Design: A total of 789 amniotic fluid single and serial values of deviations in optical density at 450 nm were performed on Rh-immunized pregnancies from 14 to 40 weeks' gestation. The relationship of the deviations in amniotic fluid values of optical density at 450 nm to varying degrees of fetal disease were examined., Results: In Rh-negative fetuses (unaffected) amniotic fluid values rise until 22 to 26 weeks before decreasing to term. In fetuses at risk of dying in utero, values are higher and trends rise. A clinical management plan was devised on the basis of the amniotic fluid findings of deviations in optical density at 450 nm., Conclusions: A clinical management scheme consisting of four zones is outlined. Rh-negative fetuses have minimal invasive procedures. Fetuses at risk of death undergo early cordocentesis for evaluation and therapy. Values that fall in between can be separated into two zones on the basis of the degree of risk.

A review of data on haemolytic disease of the newborn (HDN) collected in Newcastle upon Tyne over 25 years revealed 194 pregnancies in which anti-Kell was the only antibody detected. Sixteen affected babies were born. None was hydropic, three had very severe disease but all survived. There were also three stillbirths, none of which had post-mortem appearances of HDN. The highest recorded anti-Kell titres in individual patients ranged from 1/1 to 1/2048 and bore no relation to the severity of the disease. Of the eight pregnancies in which amniotic fluid examination predicted a high risk of stillbirth, half resulted in unaffected babies. We suggest that haemolytic disease caused by anti-Kell is less severe than suggested by some workers. The use of guidelines developed from the study of Rhesus disease to determine the need for intervention in women with anti-Kell may be inappropriate.

Three methods for the estimation of bilirubin in amniotic fluid are compared. The within-batch precision and recovery of bilirubin by each method has been studied, and the effects of the presence of haemoglobin, methaemoglobin, and methaemalbumin in varying concentrations have been investigated. Measurements on amniotic fluid from patients with rhesus isioimmunization have also been compared and regression equations calculated. The results of the three methods agree reasonably well so long as no haemoglobin degradation products are present. It is suggested that in the presence of these products a method involving organic solvent extraction of bilirubin gives the most accurate result.

Amniotic fluid a1-antitrypsin values were determined by radial immunodiffusion of 317 samples obtained between the 10th and 43rd weeks of gestation in normal pregnant patients and those with complications. Serial determinations of a1-antitrypsin values revealed that in normal patients values decreased as pregnancy advanced while values increased as pregnancy advanced in cases in which there was severe Rh-alloimmunization and hydramnios. A wide range of values was observed for each week of gestation. Although there was a definite tendency for values to decrease as pregnancy advanced (60.8 mg/100 ml before 30 weeks and 17.9/100 ml after 40 weeks); the method must be regarded as unreliable for determining fetal maturity in clinical practice. In the case of newborn infants with hyaline membrane disease, no decrease in amniotic fluid a1-antitrypsin values has been found.

Nonimmune hydrops fetalis may be associated with an elevated amniotic fluid delta OD450. Cases of I-cell disease (mucolipidosis II), lethal multiple pterygium syndrome, and alpha-thalassemia are presented, each associated with nonimmune hydrops fetalis and an elevated delta OD450. An elevated delta OD450 does not always indicate isoimmunization, and may be due to increased red blood cell turnover for a variety of reasons associated with nonimmune hydrops fetalis.

Fetal blood and amniotic fluid samples were obtained fetoscopically from 59 rhesus-isoimmunized pregnancies at 18 to 25 weeks' gestation. Fetal hemoglobin was measured and amniotic fluid optical density deviation at a wavelength of 450 nm determined. Two sets of normal reference values for optical density at 450 nm and fetal hemoglobin at 16 to 36 and 16 to 25 weeks were established from 475 amniotic fluid and 153 fetal blood samples obtained from pregnancies not complicated by fetal hemolysis. As expected, there was a significant linear correlation between the degree of fetal anemia and the amniotic fluid optical density at 450 nm in rhesus-isoimmunized pregnancies. However, the values of optical density at 450 nm were widely scattered, thereby limiting their ability to predict accurately the severity of disease in these second-trimester pregnancies. In 25 of the patients, the value of optical density at 450 nm was determined at 6 to 16 days before fetoscopy. The severity of fetal anemia could not be predicted by the trend in optical density at 450 nm. These data suggest that the only reliable method to determine the severity of rhesus isoimmunization in the second trimester of pregnancy is the direct measurement of fetal hemoglobin.

Dexamethasone or prednisolone, added in vitro to bilirubin-containing amniotic fluid, produces a time-dependent decrease in the 450-nm absorbance of the pigment. Neither the chemical determination of bilirubin in amniotic fluid nor the lecithin/sphingomyelin ratio as determined by thin-layer chromatography is affected by these glucocorticoids. The effect probably is not a result of displacement of bilirubin from its binding sites on albumin, because the absorbance of a solution of crystalline bilirubin at 450 nm is unaffected by added bovine serum albumin. Light scattering of amniotic fluid increases slightly when dexamethasone is added, whether or not low concentrations of bilirubin (less than 1.6 mumol/L) are present. Thus the effect on absorbance evidently is not ascribable to supersaturation and formation of a colloidal sol of bilirubin particles. This direct interference of glucocorticoids with the spectrophotometry of bilirubin in amniotic fluid prompts cautious interpretation of such data as an index to the severity of hemolytic disease of the fetus, specifically in cases of Rhesus-isoimmunization that are being treated with glucocorticoids.

Prediction charts used in the management of patients suffering from rhesus haemolytic disease are presented and the accuracy investigated for two methods of amniotic fluid analysis. In 468 samples from 141 patients there were 7-9 per cent errors of prediction when both methods were used together but in only four cases (2-8 per cent) was the error thought to have possibly contributed to wrong management.

The quantitation of bilirubin in amniotic fluid is of paramount importance in prognosticating the severity of Rh isoimmunization. Amniotic fluid contaminated by maternal blood can result in erroneous results when direct spectrophotemetric analysis is performed on such fluid. The technique and results of a one-step chloroform extraction performed on "bloody" amniotic fluid is presented. Results confirm that chloroform extraction yields more accurate clinical information in regard to Rh isoimmunized patients in whom a "bloody tap" is obtained.

The fluorescence polarization of 116 amniotic fluid specimens obtained from 22 isoimmunized pregnant women was determined. The degree of fluorescence polarization of amniotic fluid provides an index of microvisocity in lipid aggregates that is dependent on the lecithin-to-sphingomyelin ratio and the degree of saturation of fatty acid side chains. We confirmed the reproducibility of the measurement of amniotic fluid microviscosity (coefficient of variation, 2.0%). The measurements are not effected by bilirubin concentration of amniotic fluid dilution. The pattern of change of amniotic fluid microviscosity during gestation parallels the expected development of the surfactant system. Amniotic fluid microviscosity is high during early gestation and abruptly and sequentially decreases between the 28th and 36th week of gestation. Since the measurements are an accurate reflection of the biochemical properties of amniotic fluid lipids and parallel the development of the surfactant system, we conclude that amniotic fluid microviscosity may well serve as an indicator of the process of fetal lung maturation.

The number of Rh-D sensibilizations provided by pregnancy and delivery has decreased as the result of the preventive measures introduced to the combat this condition. The problem, however, still exists, and to cope with it, the determination of the blood group, Rh-D factor, and the Coombs' test using the amnionic fluid allows a timely preparation for the therapy of the newborn's hemolytic disease. The method of the neutralization technique employed by the authors has proved reliable.

Human placental lactogen (HPL) was analyzed in 52 samples of amniotic fluid. Assays of liquor HPL appear to aid in the evaluation of the degree of fetal involvement with erythroblastosys. The measurement of HPL in amniotic fluid may yet be proved also in the management of mothers with diabetes.

The amniotic fluids of 7 pregnant women with Rh-isoimmunization were examined. On the basis of the data of this investigation as well as of the clinical and ultrasound data intrauterine blood transfusions were made in fetuses--from 4 to 8 in number. Forty two spectrophotometric analyses were made in all, but the amniotic fluids were examined before intrauterine blood transfusions as well as before the performance of each subsequent blood transfusion. The authors found changes in the characteristic of the amniotic fluid after intrauterine blood transfusion, which were manifested by the fact that the pigment peak of delta 450 nm was reduced, but the peak of delta 410 nm was increased. In connection with these findings after intrauterine blood transfusions delta 450 nm lost its diagnostic and prognostic value. delta 410 nm before intrauterine blood transfusions manifested gravity of fetal hemolytic disease. After intrauterine blood transfusions its increase was due to blood transfusions and accumulation of methemoglobin in the amniotic fluid.

The macrophage content of amniotic fluid has been measured and the upper limit of normal on an arbitrary scale is 41. Amongst 65 amniotic fluids collected for antenatal diagnostic studies before 22weeks' gestation there were eight which had macrophage counts ranging from 82 to 6226,three of these were shown to have anencephaly and two spina bifida. The reasons for three apparently false positives are as yet undetermined. Rhesus iso-immunized amniotic fluids were found to have macrophage counts of up to 276 and a possible explanation for this is considered. It is argued that an elevated aminotic fluid macrophage count may indicate a CNS defect or possibly other fetal abnormality.

A reproducible method for the determination of bilirubin in amniotic fluid by measuring the extinction of the the chloroform extract is described. The measurements can be carried out by means of a simple spectrophotometer. In case of extraction of amniotic fluid containing blood informative results are obtained as well. Since the extinction values are equal to the deltaE-values it is possible to determine the degree of morbus haemolyticus fetalis using the schema of Liley.