Antenatal management of fetomaternal alloimmune thrombocytopenia--report of 15 affected pregnancies.

The recognition that spontaneous intracranial haemorrhage (ICH) may occur in utero in fetomaternal alloimmune thrombocytopenia (FMAIT) led us to attempt to prevent this in 15 pregnancies of 11 women who had previously affected infants with FMAIT due to anti-HPA-1a. The antenatal management included fetal platelet transfusions and maternal steroids and/or high-dose intravenous immunoglobulin (IVIgG). In the first pregnancy, ICH occurred between 32 and 35 weeks' gestation before any treatment had been given, emphasizing the need for earlier intervention. Five of the 14 subsequent pregnancies in this study were considered to be severely affected (severe haemorrhagic complications in a previous infant and initial fetal platelet count < 20 x 10(9)/L in this study); four were managed successfully with weekly fetal platelet transfusions started between 18 and 29 weeks and continued until delivery at 33-35 weeks, and one severely affected case who was referred at 36 weeks was managed successfully with a single platelet transfusion prior to delivery. Five pregnancies were considered to be mildly affected (previous infants were unaffected by severe bleeding and initial fetal platelet count > 50 x 10(9)/L in this study). The platelet counts were maintained in one case with steroids and in three with IVIgG without the need for repeated platelet transfusions, but in the fifth the fetal platelet count fell despite steroids and IVIgG and serial platelet transfusions were required. Four pregnancies were unsuccessful; two pregnancies were terminated after severe ICH occurred at an early stage before fetal blood sampling had been carried out, one fetus died after the mother had a severe fall despite the successful initiation of fetal platelet transfusions and one died due to a cord haematoma which occurred at the time of the initial fetal blood sampling. The optimal management of FMAIT to reduce the risk of antenatal ICH remains uncertain. Steroids and IVIgG may be effective in some mildly affected cases but serial fetal platelet transfusions are the preferred therapy for those who are severely affected.

Effect of the choice of WHO International Reference Preparation for thromboplastin on International Normalised Ratios.

AIMS: To compare the International Normalised Ratio (INR) obtained directly with the two types of WHO plain International Reference Preparation for thromboplastin in patients treated with coumarin. METHODS: Prothrombin times were performed in parallel at four centres using WHO human plain IRP (BCT/253) and rabbit plain IRP (RBT/79). Sixty patients and 20 normal controls were tested at each centre. Differences in INR among the centres were assessed by one factor, analysis of variance. The bias for each centre was assessed by the t test. RESULTS: At all four centres higher INRs were consistently found with the rabbit plain reagent. Two of the centres showed significantly greater bias. CONCLUSIONS: There was a small but significant difference in INR results obtained directly with these two reference reagents at all four centres (mean 7.35%). This in part may result from the different responsiveness of the two IRP to the coumarin defect or to imprecision of the original ISI calibrations of the two plain WHO IRP. The findings support the adoption of a single master IRP, in accord with WHO recommendations, which would resolve the present anomalous situation.

Molecular genetic analysis of factor X deficiency: gene deletion and germline mosaicism.

A case of homozygous factor X deficiency arising from the inheritance of two non-identical gene deletions from heterozygous parents is described. One, a partial gene deletion, was localized to exons VII and VIII by a combination of Southern blotting and polymerase chain reaction (PCR) amplification of exon sequences. The other deletion, of maternal origin, probably involves the entire factor X gene. Restriction fragments associated with the exon VII + VIII deletion were present in three siblings including the homozygous proband. These fragments were however absent from the somatic cells of the father, a finding consistent with germline mosaicism.

The molecular genetic analysis of hemophilia A: a directed search strategy for the detection of point mutations in the human factor VIII gene.

A directed-search strategy for point mutations in the factor VIII gene causing hemophilia A was used to screen eight potentially hypermutable CpG dinucleotides occurring at sites deemed to be of functional importance. Polymerase chain reaction-amplified DNA samples from 793 unrelated individuals with hemophilia A were screened by discriminant oligonucleotide hybridization. Point mutations were identified in 16 patients that were consistent with a model of 5-methylcytosine (5mC) deamination. Four new examples of recurrent mutation were demonstrated at the following codons: 336 (CGA----TGA), 372 (CGC----TGC), 372 (CGC----CAC), and 1689 (CGC----TGC). These are functionally important cleavage sites for either activated protein C or thrombin. Further novel C----T transitions were identified in the remaining arginine codons screened (-5, 427, 583, 795, and 1696), resulting in the creation of TGA termination codons. Differences in mutation frequency were found both within and between the CpG sites and between ethnic groups. These differences are assumed to be due to differences in the level of cytosine methylation at these sites, although direct evidence for this inference is lacking.

The molecular genetic analysis of haemophilia A; characterization of six partial deletions in the factor VIII gene.

In a survey of 528 unrelated haemophilia A patients, six partial deletions of the factor VIII (FVIII) gene were detected by Southern blotting. These deletions were further mapped by a combination of Southern blotting and polymerase chain reaction amplification and found to vary in length between 4.7 kb and 57 kb. The frequency of detectable FVIII gene deletions (about 1%) frequency of detectable FVIII gene deletions (about 1%) is thus considerably lower than previously reported. Statistical analysis of currently available data did not provide any evidence for a deletion "'hotspot". Four of the six deletion patients reported here possessed inhibitors. Taken together with previous data, deletion of the FVIII gene was found to be associated with an approximately five-fold higher risk of developing inhibitors compared with other severe haemophiliacs without gene deletions.

Improved carrier detection of haemophilia A using novel RFLPs at the DXS115 (767) locus.

Two novel restriction fragment length polymorphisms (RFLPs) around the DXS115 (767) locus, detectable with the restriction enzymes MspI, are described. Since DXS115 is closely linked to the factor VIII gene (F8C), the MspI RFLP was employed in haemophilia A carrier detection. The utility of these RFLPs lies in the increased applicability and accuracy of diagnoses carried out in cases where available intragenic markers are uninformative.

Management of fetal alloimmune thrombocytopenia by weekly in utero platelet transfusions.

Alloimmune neonatal thrombocytopenia (ANT) may cause intracranial haemorrhage in utero as well as at delivery. Recent management has concentrated on attempts to minimise fetal thrombocytopenia and prevent its complications. This report describes further experience with the use of repeated intravascular transfusions of compatible platelets in utero. The patient studied had already had one infant with intracranial haemorrhage due to ANT. In her next pregnancy, weekly intra-uterine platelet transfusions were given from 26 weeks, but intra-uterine death occurred at 30 weeks after the mother had a heavy fall. In her most recent pregnancy, weekly intravascular transfusions of platelets were given by cordocentesis from 29 to 34 weeks. The fetal platelet count was maintained above 30 X 10(9)/l for almost all of the last 6 weeks of pregnancy before delivery of a normal infant by Caesarean section at 35 weeks' gestation. This approach is effective in preventing severe fetal thrombocytopenia in the last trimester of pregnancy and is contrasted with alternative treatments of ANT. Further data are required to determine the efficacy and risks of these treatments.

Cordocentesis in the investigation of fetal erythropoiesis.

Fetal blood hemoglobin concentration and erythrocyte, reticulocyte, and erythroblast counts were determined in umbilical cord samples obtained from 194 pregnancies at 17 to 40 weeks' gestation. The fetuses sampled were undergoing prenatal diagnosis and were subsequently found not to be affected by the condition investigated. The hemoglobin concentration and erythrocyte count increased linearly with gestation from respective means of 11 gm/dl and 2.5 x 10(12)/L at 17 weeks to 15.5 gm/dl and 4.5 x 10(12)/L at 40 weeks. The erythroblast count decreased exponentially from a mean of 83/100 leukocytes at 17 weeks to 4/100 leukocytes at 40 weeks. The reticulocyte count decreased linearly from a mean of 27.5 x 10(9)/L or 10/100 red blood cells at 17 weeks to 17.5 x 10(9)/L or 4/100 red blood cells at 40 weeks.

Human embryonic zeta-globin chains in fetal and newborn blood.

A sensitive and specific radioimmunoassay (RIA) for human embryonic zeta-globin chains was used to study normal fetal blood and newborn cord blood as well as cord blood from newborns with alpha-thalassemias. From 17 weeks until 37 weeks of gestation, zeta-globin chains were present in almost all fetal and cord blood samples (0.27% +/- 0.15% in samples of weeks 17 through 30; 0.14% +/- 0.11% in samples of weeks 31 through 37). zeta-Globin chains were present in greater than 80% of cord blood hemolysates from normal, full-term newborns (0.15% +/- 0.11%) as well as from 16 near-term newborns of diabetic mothers (0.13% +/- 0.13%). zeta-Globin chains were not detected in normal infants aged 3 months to 2 years. In cord blood hemolysates from alpha-thalassemic newborns, the levels of zeta-globin chain content varied from very high to undetectable levels. Gene mapping of the zeta-alpha-globin gene cluster was performed in 12 newborns in whom cord blood zeta-globin chains had been determined. Newborns who were carriers of alpha-thalassemia-1 due to the (--SEA/) deletion had very high levels of zeta-globin chains (greater than 1.5%).

Molecular pathology of haemophilia B.

Direct sequencing of amplified genomic DNA has been used to investigate the molecular basis of haemophilia B and thus identify specific amino acids that are essential for maintenance of structure or function of factor IX. Substitution of Cys 336, Asn 120 results in loss of circulating factor IX antigen and deletion of Arg 37 in gross reduction of circulating protein and loss of activity, while substitution of Arg -4, Arg 333, Asp 64 and Pro 55 cause loss of function without marked reduction in protein serum levels. Frameshift or point mutations resulting in marked loss of coding information are found in patients who develop antibodies to administered factor IX. An enhanced rate of mutation is evident at two CpG dinucleotides in the factor IX gene, which accounts for approximately 25% of all point mutations causing haemophilia B known to date. Direct sequencing of mutations also permits, for the first time, rapid and unequivocal prenatal and carrier diagnoses, in all cases, by eliminating the need for informative segregation of markers.

Prenatal diagnosis of haemoglobinopathies by ion exchange HPLC of haemoglobins.

A new method, the cation exchange HPLC of haemoglobins, has been compared to the classical carboxymethyl cellulose (CMC) chromatography of globin chains for the prenatal diagnosis of beta thalassaemia and sickle cell disease. The two methods correlated highly. The HPLC procedure can use two independent and reliable means--optical density at 405 nm and radioactivity to determine the adult Hb/HbF+Fac ratio. The diagnosis is obtained in 15 min by cation exchange HPLC.

Erythroblastosis and reticulocytosis in anemic fetuses.

The fetal blood erythroblast and reticulocyte counts were determined in umbilical cord samples obtained at 17 to 36 weeks' gestation from 127 pregnancies complicated by red blood cell isoimmunization. The reticulocyte count increased linearly with fetal anemia, and the erythroblast count increased exponentially. Significant erythroblastosis was observed only when the hemoglobin concentration deficit was greater than 7 gm/dl. Of the 52 fetuses with a hemoglobin concentration deficit greater than 7 gm/dl, 35 had ultrasonographic evidence of hydrops. These data suggest that medullary hematopoiesis is stimulated by mild anemia and that recruitment of extramedullary sites occurs when anemia is severe. Extensive hepatic erythropoiesis may be the cause of fetal hydrops in red blood cell isoimmunization.

A factor IX mutation, verified by direct genomic sequencing, causes haemophilia B by a novel mechanism.

A novel factor IX gene mutation (factor IX London 2) has been characterized. This causes severe crm+ haemophilia B as the patient's plasma shows normal factor IX antigen level and less than 1% clotting activity. Sequence analysis of the entire cloned coding and promoter regions revealed a single point mutation: a G----A transition at position 31,119. This region of the patient's DNA was amplified in vitro by the polymerase chain reaction and the nucleotide change was confirmed by direct sequencing of the amplified products. The mutation results in the substitution of the arginine at position 333 by glutamine. This arginine residue is absolutely conserved in the catalytic domain of normal human and bovine factor IX, X and prothrombin. The substitution by glutamine causes the loss of a positive charge from the surface of the factor IX London 2 protein. This mutation pinpoints a previously unknown, functionally critical feature of factor IX which may be involved in substrate or co-factor binding.

Fetal haemoglobin measurement in the assessment of red cell isoimmunisation.

A reference range of fetal haemoglobin concentration (g/dl) was established from umbilical cord blood samples obtained by cordocentesis (n = 200) or at delivery (n = 10). In normal pregnancy the mean fetal haemoglobin increases linearly from 11 g/dl at 17 weeks' gestation to 15 g/dl at 40 weeks' gestation and one standard deviation is approximately 1 g/dl. The haemoglobin was also measured in fetal blood from 154 red cell isoimmunised pregnancies from 17 to 36 weeks' gestation. In 48 fetuses with ultrasound features of hydrops the haemoglobin was 7-10 g/dl below the normal mean for gestation. It is proposed that in pregnancies complicated by red cell isoimmunisation the severity of the disease should be assessed and treated on the basis of the deviation of the fetal haemoglobin from the normal mean for gestation into mild (haemoglobin deficit less than 2 g/dl), moderate (deficit 2-7 g/dl), and severe (deficit greater than 7 g/dl).

Partial deletion by illegitimate recombination of the factor IX gene in a haemophilia B family with two inhibitor patients.

The inhibitor phenotype occurs in six haemophilia B patients in the UK and results from development of antibodies by the patients to administered factor IX. We have analysed a partial factor IX gene deletion (London 1) in a family with two inhibitor patients. The deletion results in retention of the first five exons which code for the light chain of factor IXa, and removal of 23 kb of DNA starting 704 bp 3' of the fifth exon and terminating 10.3 kb 3' of the last exon. The 5' break is at residue -113 of an Alu repeat. No significant homology exists between the 5' and 3' termini, but a 9 bp region of complementarity is found 23 bp and 60 bp from the 5' and 3' terminus, respectively. At the cloned deletion junction a new 16 bp sequence contributes a DraI site that is also found in the genomic DNA of the two patients and a heterozygous relative. The deletion is an example of illegitimate recombination and it is proposed that such deletions occur principally during DNA replication. Loss of the 3' sequences involved in the maturation of mRNA probably results in no factor IX production. Immunological studies show that the index patient's antibodies bind both to epitopes coded by deleted and by non-deleted segments of the gene.

Diagnosis and treatment of feto-maternal hemorrhage in a fetus with homozygous von Willebrand's disease.

In a fetus with severe type-III von Willebrand's disease, fetal blood sampling by cordocentesis was associated with feto-maternal hemorrhage, fetal hypovolemia, and persistent bradycardia. The fetal condition improved after intracardiac transfusion of blood.