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PubMed1986

Use of the erythrocyte rosette test to screen for excessive fetomaternal hemorrhage in Rh-negative women.

The possibility of Rh immune globulin failure exists when a fetomaternal hemorrhage exceeds 25 to 30 ml of whole blood and only one 300 micrograms vial of Rh immune globulin is administered. In this prospective study of 1000 consecutive Rh-negative women who were delivered of Rh-positive newborn infants, the presence of fetal erythrocytes in maternal blood was identified with use of both the Du test read microscopically and the erythrocyte rosette test. All positive tests prompted fetomaternal hemorrhage quantification with use of a modified Kleihauer-Betke acid elution test. Nineteen patients demonstrated a positive rosette test, and the only positive Du tests were in five of these 19. Six of the nineteen had levels of greater than 30 ml of whole blood for an incidence of 0.6% for fetomaternal hemorrhage exceeding the protective capabilities of the standard Rh immune globulin dosage. In experiments with simulated fetomaternal hemorrhage, all 79 samples, containing from 2.5 to 70 ml of fetal whole blood, were positive according to the erythrocyte rosette test. Applying the Du test to the same samples resulted in a 30% false negative rate at the level of a 30 ml simulated hemorrhage. Based on sufficient sensitivity, ease of interpretation, and reasonable cost, the rosette test appears to be a superior screening test for excessive fetomaternal hemorrhage in Rh immune globulin candidates.

Blood Volume Determination↗
PubMed1987

Clinical high-risk designation does not predict excess fetal-maternal hemorrhage.

During a 5-year period, an enzyme-linked antiglobulin test was used to screen and quantitate fetal-maternal hemorrhage in 789 consecutive D-negative mothers who were delivered of D-positive babies. Six hundred seventy-two patients (85.2%) had no detectable fetal-maternal hemorrhage, and 117 patients (14.8%) had a detectable fetal-maternal hemorrhage. Eight of the 789 (1%) had a fetal-maternal hemorrhage greater than 30 ml and required more than one vial of Rh immune globulin. Two patients with fetal-maternal hemorrhage of 29 and 30 ml also received additional Rh immune globulin. Each case was reviewed for the presence of high-risk features that are thought to predict patients at risk for fetal-maternal hemorrhage. Patients having a cesarean section or complicated vaginal delivery were considered to be in a group at high risk for fetal-maternal hemorrhage, while those with a spontaneous vaginal delivery were considered not to be at risk for fetal-maternal hemorrhage. Thirty-two of 237 patients (13.5%) in the risk group and 82 of 552 patients (15.3%) in the group not at risk had detectable fetal-maternal hemorrhage. The incidence of fetal-maternal hemorrhage for these two groups was not statistically different (p greater than 0.50 by chi 2 analysis). If only patients in the risk group had been screened for fetal-maternal hemorrhage, then five of 10 (50%) who required more than one vial of Rh immune globulin would have been undertreated and at risk for developing anti-D antibodies. In addition, newborn birth weight, Apgar scores, and hematocrits were examined for 13 cases of fetal-maternal hemorrhage of greater than or equal to 21 ml, and none of these characteristics could be used to predict patients at risk for fetal-maternal hemorrhage. Therefore, no maternal or newborn characteristics could be identified that would reliably predict patients at risk for fetal-maternal hemorrhage. We conclude that all D-negative patients with D-positive babies should continue to be screened for fetal-maternal hemorrhage to identify those patients requiring more than one vial of Rh immune globulin.

Delivery, Obstetric↗
PubMed1990

The incidence of significant fetomaternal hemorrhage in patients undergoing cesarean section.

To determine the incidence of fetomaternal hemorrhage in patients undergoing cesarean section, Kleihauer-Betke tests were performed in the immediate postoperative period on 199 parturients. Some degree of hemorrhage was detected in 18.5% of patients, with 2.5% demonstrating greater than 30 ml of fetal blood. Comparison of groups on the basis of indication for cesarean delivery revealed no difference in rates of fetal hemorrhage. Because patients with greater than 30 ml of fetal blood would not be adequately protected from Rh sensitization by the standard 300 micrograms dose of Rh immune globulin, we recommend screening all Rh-negative patients undergoing cesarean section for the presence of significant fetomaternal hemorrhage.

Cesarean Section↗
PubMed1994

The use of polymerase chain reaction to determine fetal RhD status.

OBJECTIVE: Our purpose was (1) to establish the accuracy of a deoxyribonucleic acid amplification method in determination of RhD status in adult blood samples, including weak D variants (previously referred to as Du) and a D mosaic, and (2) to apply the method to determine fetal RhD status in alloimmunized pregnancies. STUDY DESIGN: Twenty-five adult blood samples, including five weak D variants and one D mosaic, were analyzed with a polymerase chain reaction to determine RhD type. The method was then applied to amniotic fluid samples obtained by amniocentesis from three RhD-negative women with known RhD sensitization. RESULTS: RhD type determined by polymerase chain reaction for all adult blood samples agreed with serologic typing results. All weak D variants and the D mosaic gave results consistent with RhD positivity. Fetal RhD status was determined in each of the three alloimmunized pregnancies, and obstetric management decisions were made on the basis of these results. CONCLUSIONS: This polymerase chain reaction method allows rapid and accurate determinations of fetal RhD status by amniocentesis. Fetal blood sampling or serial amniocenteses may be avoided when the fetus is RhD negative, and plans for surveillance and intervention can be confidently made if the fetus is RhD positive. However, before the widespread use of this assay, its sensitivity and specificity must be established. Because weak D variants and a D mosaic demonstrated RhD-positive status by polymerase chain reaction, the method described is applicable to these RhD variants.

Adult↗
PubMed1995

Accuracy of prenatal determination of RhD type status by polymerase chain reaction with amniotic cells.

OBJECTIVE: Our purpose was to determine the accuracy of RhD typing by use of amniocytes obtained at amniocentesis in RhD-negative women. STUDY DESIGN: One hundred thirty-five RhD-negative women undergoing amniocentesis for management of suspected alloimmunization (n = 95) or routine second-trimester cytogenetic indications (n = 40) were studied. Amniocytes were then used as template to amplify specific portions of the Rh D and Rh CcEe genes by polymerase chain reaction. The fetal RhD type was confirmed by serologic techniques either after fetal blood sampling or cord blood samples at birth. RESULTS: Thirty-six fetuses were serologically typed as RhD negative and all 36 were typed RhD negative by polymerase chain reaction. Ninety-eight fetuses were serologically typed as RhD positive; of these, 96 were correctly typed as RhD positive and two were incorrectly typed as RhD negative, with an overall error rate of 1.4%. Both of the errors occurred in a single batch of six samples tested at the same time. In one of these cases the fetus had mild Rh alloimmune disease and required exchange transfusion at birth. In the second case the fetus had severe hydrops fetalis and died in utero at 28 weeks. Deoxyribonucleic acid isolated from fetal blood was tested with the same polymerase chain reaction technique after delivery, and in both cases the fetus was correctly typed as RhD positive. Deoxyribonucleic acid amplification repeatedly failed in one case. CONCLUSION: Prenatal fetal RhD typing by polymerase chain reaction with amniotic fluid cells is accurate and reliable. In sensitized pregnancies it allows early management of Rh disease and avoids invasive procedures in RhD-negative fetuses. In nonsensitized pregnancies it avoids the use of anti-RhD immunoglobulin after invasive procedures or during pregnancy. To eliminate the possibility of genetic and laboratory sources of errors, we suggest using different sets of primers at two different loci (e.g., exon 4 to 5 and exon 10).

Amniocentesis↗
PubMed1979

Immunoprophylactic anti-Rho(D) treatment after mismatched transfusions.

80 cases of anti-Rho(D) treatment after mismatched transfusions are reviewed to compare the intramuscular (i.m.) and intravenous (i.v.) route of treatment. No severe reactions such as renal failure occurred with either method of anti-D treatment. If the Rh-immune globulin is injected i.m., 20 micrograms anti-D per ml red cells are used. For i.v. anti-D administration, 10-12 micrograms anti-D are suggested. The i.v. method of anti-D treatment is recommended for the future. Furthermore, a scheme of treatment for after mismatched transfusions is described.

Blood Grouping and Crossmatching↗
PubMed1995

Polymerase chain reaction determination of RhD blood type: an evaluation of accuracy.

OBJECTIVE: To evaluate the accuracy of polymerase chain reaction (PCR) amplification of a portion of the RhD gene by testing a large number of DNA samples derived from individuals whose RhD status was established by the standard serologic method. METHODS: Seven hundred sixty-five samples were obtained from two sources: subjects taking part in studies at the University of Iowa Hospitals and Clinics (n = 107), and Centre d'Etude du Polymorphisme Humain (CEPH) families used for studies of genetic variation (n = 658). Deoxyribonucleic acid was extracted from blood samples of University of Iowa volunteers and from CEPH families by standard techniques. With few modifications, published primers, reaction and electrophoresis conditions, which yield a 1200-bp fragment in all individuals and a 600-bp fragment in RhD-positive individuals, were used. RESULTS: By standard serologic techniques, we identified samples from 632 RhD-positive and 133 RhD-negative individuals. Two (both from CEPH) of the 632 RhD-positive individuals were characterized as RhD-negative by PCR. Seven of the 133 RhD-negative samples were judged to be RhD-positive by PCR because of the presence of a light 600-bp band. Despite repeated attempts, no bands or DNA were identified in one RhD-negative sample. Thus, the sensitivity of RhD typing by PCR was 99.7%, the specificity 94.0%. CONCLUSION: Based on our data, it would appear that the use of PCR to establish RhD type can be introduced cautiously into current management schemes in the evaluation of RhD sensitization.

Blood Grouping and Crossmatching↗
PubMed1987

The evaluation of maternal anti-D concentrations during pregnancy.

Anti-D quantitation by the AutoAnalyzer technique has been shown to be a helpful aid in assessing the severity of D alloimmunization during pregnancy. In this study, the technique has been used both to detect antibody boosting after amniocentesis and to differentiate active D immunization from the presence of passive antibodies. The AutoAnalyzer technique and the more generally used indirect antiglobulin test titration method showed good agreement at titre levels of 32 or lower. A titre of 32 was found to be a good discriminative level to separate the mildly affected from the more severely affected newborns suffering from Rh haemolytic disease. At higher titre levels, however, the AutoAnalyzer technique was the method of choice for correct clinical assessment of the severity of D alloimmunization.

Amniocentesis↗
PubMed1987

A management programme for Rh alloimmunization during pregnancy.

A management programme for the control and treatment of Rh0 (D) immunized during pregnancy is presented. A total of 34,650 births were registered during a 4.5 year period and included 63 D positive newborns to D-immunized mothers. The outcome of all infants has been evaluated according to the severity of the haemolytic disease. Exchange transfusion was unnecessary in 43 mild cases (68.3%). Fourteen infants (22.2%) required exchange transfusion, and in 6 severe cases (9.5%) maternal plasma exchange and exchange transfusion was performed. No detrimental effects or deaths occurred among the infants suffering from Rh haemolytic disease. We recommend that the frequency and volume of plasma exchange therapy should be individually adjusted to suit each patient and the effect monitored regularly through maternal anti-D levels using a sensitive quantitative technique.

Amniotic Fluid↗
PubMed1995

Are there options for donor-derived i.m. anti-D IgG preparations other than to prevent Rh(D) sensitization? The intravenous route.

The use of i.v. anti-Rh(D) IgG for conditions other than prevention of Rh(D) sensitization is discussed. Besides highlighting the platelet response in ATP, a putative distinctive effect on the hemorrhagic threshold is suggested. Accordingly, we have included discussion of cases of aplastic anemia, myelodysplasia, heavy chemotherapy, coagulation deficiency, and senile vascular atrophy. We have also considered attempts to replace the high-dose pooled i.v. IgG (IVIG) with the much smaller amounts of IgG present in anti-Rh(D) preparations used to prevent Rh-sensitization in pregnancy. Both Fc and variable fragments of the IgG molecule may play a role, the former potentiated by manufacture-induced IgG aggregation and the latter by donor hypersensitization. A role for IgG-anti-F(ab')2 molecules cannot be ruled out.

Blood Donors↗
PubMed2005

Fetal RhD genotyping by maternal serum analysis: a two-year experience.

OBJECTIVE: The purpose of this study was to determine the accuracy of the none-invasive prenatal determination of polymerase chain reaction (PCR)-based fetal RhD genotyping. STUDY DESIGN: A prospective case series was undertaken on all RhD-negative pregnant women presenting for genetic counseling in our prenatal diagnosis center from January 2001 until December 2002. Results were compared with serologic RhD typing of the newborns. RESULTS: Among the 285 pregnant women who participated in the study, fetal RhD status could be determined for 283 patients. In 2 cases, the RhD-negative phenotype of the mother was not the result of a complete RHD gene deletion, and therefore, the status of the fetus could not be determined. Neither false-negative nor false-positive results were observed. CONCLUSION: The present report demonstrates that a reliable fetal RHD genotype determination can be achieved with 100% accuracy. It is therefore possible to consider that such an assay could be systematically proposed to all RhD-negative pregnant women in order to more effectively utilize RhD prophylaxis.

Blood Grouping and Crossmatching↗
PubMed2006

Diagnostic accuracy of noninvasive fetal Rh genotyping from maternal blood--a meta-analysis.

OBJECTIVE: The purpose of this study was to determine the reported diagnostic accuracy, the validity, and the current limitations of fetal Rh genotyping from peripheral maternal blood based on the existing English-written publications. STUDY DESIGN: A search of the English literature describing fetal RhD determination from maternal blood was conducted. From each study, we determined the number of samples tested, fetal RhD genotype, the source of the fetal DNA (maternal plasma, serum, or fetal cells), gestational age, and confirmation of fetal Rh type. The presence of alloimmunization and exclusions of tested samples were noted. For the meta-analysis we calculated composite estimates using 2 random effects models, weighted GLM and Bayesian. Sensitivity, specificity, positive and negative predictive values were calculated. RESULTS: We identified 37 English-written publications that included 44 protocols reporting noninvasive Rh genotyping using fetal DNA obtained from maternal blood on a total of 3261 samples. A total of 183 (183/3261, 5.6%) samples were excluded from the meta-analysis. The overall diagnostic accuracy after exclusions was 94.8%. The gestational ages ranged between 8 and 42 weeks gestation. Maternal serum and plasma were found to be the best source for accurate diagnosis of fetal RhD type in 394/410 (96.1%) and 2293/2377 (96.5%), respectively. There were 719/783 (91.8%) alloimmunized patients that were correctly diagnosed. There were 16 studies that reported 100% diagnostic accuracy in their fetal RhD genotyping. CONCLUSION: The diagnostic accuracy of noninvasive fetal Rh determination using maternal peripheral blood is 94.8%. Its use can be applicable to Rh prophylaxis and to the management of Rh alloimmunized pregnancies. Improvements of the technique and further study of structure and rearrangements of the RhD gene may improve accuracy of testing and enable large-scale, risk-free fetal RhD genotyping using maternal blood.

DNA↗