The reversible antithrombin activity of incubated plasma.

The thrombin time of normal citrated plasma is progressively prolonged on incubation in open glass tubes at 37 degrees C. The phenomenon is dependent on the temperature and duration of incubation, on the pH, and on the concentration of calcium ions present. Platelet-rich citrated plasma fails to exhibit augmented antithrombin activity when similarly incubated, and the addition of washed platelets to platelet-poor plasma inhibits this activity. The clot retarding action of incubated plasma against thrombin is also manifested against Arvin (Ancrod), but not against Reptilase-R. This thrombin time lengthening may be inhibited by incubation with anti-antithrombin III antiserum thus indicating that the phenomenon of thrombin time lengthening is consistent with enhanced activity of antithrombin III. It is unlikely that alterations in the activity of alpha2-macroglobulin, is important in the reduced thrombin-coagulability of incubated plasma. Interference with the polymerisation of fibrin monomers by the physico-chemical changes may contribute to the observed phenomenon.

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.

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.

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.

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.

Prenatal diagnosis of beta thalassaemia by reverse phase HPLC.

Reverse phase HPLC of radioactive globin chains has been compared to classical carboxy methyl cellulose chromatography for the prenatal diagnosis of beta thalassaemia. The two methods correlated highly (r = 0.97 p less than 0.0005) and provided an identical diagnosis for 40 fetal blood samples of fetuses homozygous or heterozygous for beta thalassaemia. The HPLC procedure was much faster and required fewer biochemical steps (no globin preparation). It was at least as accurate and more sensitive than the classical chromatography. A single column can be used for 150 analyses and is always ready to be used. Last but not least it is much less expensive than CMC chromatography.

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.

Fetal haematology in rhesus isoimmunisation.

Haematological studies were carried out in pure fetal blood samples obtained fetoscopically in 29 rhesus isoimmunised pregnancies at 18-24 weeks' gestation, and the values were compared with those obtained in 62 normal control pregnancies. Fetal reticulocytosis or erythroblastaemia was seen only in association with a haemoglobin concentration of 4 g/dl or less. Ten of the 14 fetuses with a haemoglobin concentration below 4 g/dl showed ultrasonographic evidence of hydrops.

Selective feticide of the affected twin by fetoscopic air embolism.

Three twin pregnancies, each with one affected fetus (by microcephaly, haemophilia A and spina bifida respectively) were encountered. Selective feticide was performed at the patient's request by injection of filtered air into an umbilical vessel by fetoscopy. The method was successful in all three pregnancies two of which resulted in a live, healthy baby.

Have Liley charts outlived their usefulness?

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.

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.

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).

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.

Normal blood cell values in the early mid-trimester fetus.

Samples of pure fetal blood from 116 fetuses of 15-21 weeks' gestation were obtained by direct vision fetoscopy. Ninety nine of these fetuses, presumed to be haematologically normal, were suitable for analysis. The data obtained show that the erythropoietic system is evolving rapidly in this gestational age range. The myeloid series shows no significant increase or decrease in numbers apart from eosinophils and basophils which increase significantly with gestational age whereas the platelet count remains constant. The growing application of fetoscopic blood sampling to the prenatal diagnosis and management of fetal blood disorders renders mandatory a knowledge of normal fetal blood values.

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%).

Fetoscopy in the assessment of unexplained fetal hydrops.

Pure fetal blood samples, obtained fetoscopically from 30 patients with unexplained fetal hydrops at 16 to 32 weeks gestation were investigated for cytogenetic, haematological, biochemical and virological properties. In two patients with oligohydramnios, the fetoscope was introduced transabdominally into the fetal peritoneal cavity and sampling was undertaken from the intra-abdominal portion of the umbilical vein; in all the other patients an umbilical cord vessel was sampled. Ten (33%) of the fetuses had chromosomal abnormalities, one an erythroblastic process, possibly erythroleukaemia, one alpha-thalassaemia and one cytomegalovirus infection. Blood-film abnormalities were seen in 23 (88%) of 26 fetuses that had this examination. Biochemical analysis of fetal plasma was undertaken in 18 fetuses and hypoproteinaemia was found in all cases. One fetus was subsequently found to have a paroxysmal tachyarrhythmia that responded to digitilization. Three (10%) of the fetuses survived.

Plasma assay of fetal factors VIIIC and IX for prenatal diagnosis of haemophilia.

Fetal blood unmixed with maternal blood or amniotic fluid was obtained by direct-vision fetoscopy in 22 consecutive cases at 15--22 weeks' gestation; the investigation was done either for prenatal diagnosis or before therapeutic abortion. Fetal plasma factors VIIIC and IX averaged 50 I.U./dl (S.D. 12.8) and 12.5 I.U./dl (S.D. 2.4), respectively. Two male fetuses at risk of haemophilia had normal factor VIIIC levels by these criteria, and both pregnancies ended in the birth of a normal boy. Five others gave 3 normal and 2 haemophilic results, which were confirmed in two of the three terminated pregnancies.