Abnormal activated partial thromboplastin time and malignancy.

Malignancy often results in clotting abnormalities. The aetiology of haemostasis problems in cancer is complex, and is still not completely understood. We describe a case of a patient with malignant mesothelioma, who was found to have elevated activated partial thromboplastin time, due to lupus anticoagulant. We suggest that patients with malignancy should have their coagulation checked prior to any invasive procedures.

Severe gestational (incidental) thrombocytopenia: to treat or not to treat.

Thrombocytopenia is common in pregnancy and is diverse in etiology. Immune thrombocytopenic purpura (ITP) may affect both mother and the newborn. Gestational (incidental) thrombocytopenia in an otherwise fit woman, at term is the most frequent type of thrombocytopenia and poses no clinical consequences for mother or infant. We report six women who presented with severe thrombocytopenia during pregnancy. Five were treated in late pregnancy, either with intravenous immunoglobulin (IVIg), or IVIg followed by steroids. There was no response, and four received a platelet transfusion during delivery. The platelet counts in all the infants were normal and the maternal thrombocytopenia resolved spontaneously after delivery in all cases. Our observations suggest that this is a group of patients with a severe form of gestational thrombocytopenia. The severe form of gestational thrombocytopenia appears to be rare, and recognition is important, as it may recur in subsequent pregnancies and does not require any therapeutic intervention.

Fetal and embryonic hemoglobins in erythroblasts from fetal blood and fetal cells enriched from maternal blood in pregnancies complicated by maternal diabetes mellitus.

OBJECTIVES: To determine whether there is a delay or reversal in switch mechanisms from embryonic (epsilon and zeta) to fetal (gamma) hemoglobins accompanying the erythroblastosis in fetuses of diabetic mothers, and whether the increased erythroblast count in the fetal blood is associated with an increase in fetomaternal cell trafficking. MATERIALS AND METHODS: Fetal and maternal blood samples were obtained from 11 fetuses and five pregnant women in pregnancies complicated by maternal diabetes mellitus. Blood samples were also taken as controls from 35 fetuses and 33 mothers. Fetal erythroblasts were isolated by triple density gradient centrifugation and magnetic cell sorting with anti-CD71 antibody. Fluorescent antibodies were used to immunostain for gamma (gamma), epsilon (epsilon) and zeta (zeta) hemoglobin chains. In the maternal samples, fluorescence in situ hybridization for X and Y chromosomes was also carried out, to confirm the presence and proportion of enriched fetal cells from the maternal blood. RESULTS: In both fetal and maternal blood the median percentages of erythroblasts positive for gamma-globin, epsilon-globin and zeta-globin chains were significantly higher in fetuses of diabetic mothers compared to controls (fetus, gamma-globin, 76 vs. 64%, p < 0.0001; epsilon-globin, 4 vs. 0%, p < 0.0001; zeta-globin, 4 vs. 0%; p < 0.0001; mother, gamma-globin, 14 vs. 1%, p < 0.0005; epsilon-globin, 0.25 vs. 0%, p < 0.0003; zeta-globin, 0.2 vs. 0%, p < 0.0003). The median percentage of cells with Y signals in maternal blood was also higher in diabetic pregnancies compared to normal controls (7.5 vs. 1%, p < 0.002). CONCLUSIONS: The findings suggest that the fetal erythroblastosis in diabetic pregnancies is accompanied by a delay in the switch from embryonic to fetal hemoglobin chains. In addition, it is associated with an increase in fetomaternal cell trafficking.

Assessment of efficacy of cell separation techniques used in the enrichment of foetal erythroblasts from maternal blood: triple density gradient vs. single density gradient.

The aim of this study was to determine the efficacy of cell separation with single density and triple density-gradient techniques in the yield of foetal erythroblasts isolated from maternal blood. Maternal blood was obtained from 20 singleton pregnancies at 11-14 weeks of gestation immediately before foetal karyotyping by chorionic villus sampling. In each woman, the blood sample was divided into two portions; one portion was used for single density-gradient separation and the other, for triple density-gradient separation. Magnetic cell sorting (MACS) was subsequently performed with anti-CD71/antiglycophorin-A. The enriched erythroblasts were stained with Kleihauer-Giemsa and with fluorescent antibodies for the gamma, epsilon and zeta globin chains. The percentage of foetal cells positive for each stain was calculated. Fluorescence in situ hybridization (FISH) for X- and Y-chromosomes was also performed. Comparison was made in the proportion of enriched foetal cells between the two separation methods for each CD71 and glycophorin-A (GPA) antibody. The percentage of erythroblasts enriched from maternal blood that stained positive for gamma, epsilon and zeta globin chains and with Kleihauer-Giemsa was significantly higher in the triple density-gradient separation fractions compared with the single density-gradient fractions with both anti-CD71 and GPA MACS. FISH analysis for the Y-chromosome confirmed the increase in foetal cell proportion in the triple density-gradient samples. Isolation of foetal erythroblasts from maternal blood using triple density-gradient separation and MACS is more effective with regard to foetal cell yield and purity than single density-gradient separation and MACS.

Fetal erythroblasts in maternal blood in relation to gestational age.

OBJECTIVES: To determine the distribution of fetal erythroblasts in the maternal circulation at different gestations. MATERIALS AND METHODS: Maternal blood was obtained from 152 normal singleton pregnancies at 11-40 weeks of gestation. Fetal erythroblasts were isolated using triple density gradient separation and anti-CD71 magnetic cell sorting techniques. The enriched erythroblasts were stained with Kleihauer-Giemsa and with fluorescent antibodies for the zeta (zeta), epsilon (epsilon) and gamma (gamma) globin chains. The percentage of fetal cells positive for each stain was calculated. Fluorescence in situ hybridization for X and Y chromosomes was also performed. Comparison was made in the proportion of positive fetal erythroblasts among the different gestational ages. RESULTS: The proportion of erythroblasts stained positive with gamma-globin chain and Kleihauer-Giemsa decreased with gestation from a median of 2% at 11 weeks to 0.5% at 40 weeks. Similarly, there was a decrease in the percentage of Y-signal-positive cells from 1% at 11 weeks to 0.3% at 40 weeks. The proportion of enriched fetal erythroblasts stained positive with zeta- and epsilon-globin chains decreased exponentially from respective medians of 0.6% and 1.5% at 11 weeks to zero after 19 weeks and 24 weeks. CONCLUSION: In normal singleton pregnancy the percentage of fetal erythroblasts enriched from maternal blood decreases with gestation.

Treatment requirements of infants with rhesus isoimmunisation within a geographically defined area.

OBJECTIVE: To provide population based data on the treatment requirements of infants with rhesus isoimmunisation. SETTING: Twenty nine hospitals in South Thames in which 81 119 deliveries occurred between February 1999 and January 2000. DESIGN: Every month, a clinician identified in each of the hospitals sent back a postcard indicating whether or not an infant with RhD had required treatment in their institution. Antenatal and postnatal information was then requested from all those who gave positive responses. MAIN OUTCOME MEASURES: Requirement for postnatal treatment for rhesus isoimmunisation. RESULTS: During the one year study period, only 26 infants required treatment for rhesus isoimmunisation. The median duration of phototherapy of the 26 infants was five days (range 1-12). Seven infants required at least one exchange transfusion (two required two exchange transfusions), and seven infants received one "top up" transfusion. None received erythropoietin and no infant died. CONCLUSION: The results suggest that few infants require treatment for rhesus isoimmunisation.

Fetal and embryonic hemoglobins in erythroblasts from fetal blood and fetal cells enriched from maternal blood in fetal anemia.

BACKGROUND AND OBJECTIVES: To determine whether there is a delay or reversal in switch mechanisms from embryonic (e and z) to fetal (g) hemoglobins accompanying the erythroblastosis of anemic fetuses and whether an increased erythroblast count in fetal blood is associated with an increase in feto-maternal cell trafficking. DESIGN AND METHODS: Fetal and maternal blood samples were obtained from 10 cases with rhesus isoimmunization and 2 cases with maternal Parvo-B19 virus at 19-33 weeks' gestation. Blood samples were also taken as controls from 61 fetuses and 86 mothers. Fetal erythroblasts were isolated by triple density gradient centrifugation and magnetic cell sorting with CD71 antibody. Fluorescent antibodies were used to immuno-stain for zeta (z), epsilon (e) and gamma (g) hemoglobin chains. In the maternal samples, fluorescence in situ hybridization (FISH) for X and Y chromosomes was also carried out to confirm the presence and proportion of the enriched fetal cells from maternal blood. RESULTS: In both fetal and maternal blood the percentage of erythroblasts positive for g-globin chain was significantly higher in the anemic fetuses compared to the controls (fetal blood, p<0.001, R=0.91; maternal blood, p<0.001, R=0.56), but there was no significant difference in expression of the e and z-chains. The percentage of cells with Y-signals was also higher in the maternal samples of anemic fetuses compared to normal controls (p<0.001, R=0.56). INTERPRETATION AND CONCLUSIONS: These findings suggest that the erythroblastosis of anemic fetuses is not accompanied by a delay or a reversal in switch from embryonic to fetal hemoglobin chains. Severe fetal anemia is associated with an increase in feto-maternal cell trafficking.

Distribution of fetal and embryonic hemoglobins in fetal erythroblasts enriched from maternal blood.

BACKGROUND AND OBJECTIVES: To determine the distribution of embryonic and fetal hemoglobin chains in fetal erythroblasts isolated from maternal blood in the first trimester of pregnancy and establish the feasibility of using these chains as markers for fetal cell identification. DESIGN AND METHODS: Maternal blood was obtained from 187 singleton pregnancies at 11-14 weeks of gestation immediately before fetal karyotyping by chorionic villus sampling. In all cases included in this study the fetal karyotype was normal. Fetal erythroblasts were isolated using triple density gradient separation and anti-CD71 magnetic cell sorting techniques. The enriched erythroblasts were stained with Kleihauer-Giemsa and with fluorescent antibodies for the zeta (z), epsilon (e) and gamma (g) globin chains. The percentage of fetal cells positive for each stain was calculated. Fluorescent in situ hybridization (FISH) for X and Y chromosomes was also performed. Comparison was made with the percentage of cells with positive Y-signal FISH in pregnancies with male fetuses. RESULTS: The percentage of fetal erythroblasts stained positive was 37% for the z and 95% for both e and g globin chains, as well as the Kleihauer-Giemsa staining. There was a significant association between the Kleihauer-Giemsa stained cells and those stained with e and g globin chains. There was also an association between cells with Y-signals and those stained with e and g globin chains. INTERPRETATION AND CONCLUSIONS: Embryonic hemoglobin chains can be detected in the enriched fetal erythroblasts, with higher percentages of the e rather than the z globin chains. These chains are therefore potentially unique markers to be used in the identification of cells of fetal origin from maternal blood for prenatal diagnosis of genetic and chromosomal abnormalities.

Transfusion-related acute lung injury associated with interdonor incompatibility for the neutrophil-specific antigen HNA-1a.

BACKGROUND AND OBJECTIVES: A patient transfused with two pooled platelet concentrates became breathless. Bilateral infiltrates were seen on chest X-ray. A diagnosis of transfusion-related acute lung injury (TRALI) was made. The patient received 100% oxygen and recovered after 5 days. MATERIALS AND METHODS: Antibody screening, cross-matching for granulocyte and lymphocyte antibodies and typing for granulocyte antigens was undertaken. RESULTS: The patient typed as HNA-1b/HNA-1b. Granulocyte and lymphocyte antibodies were not detected in the patient's serum or in any of the donor sera by cross-match. In antibody screening against typed panel granulocytes, complement-fixing anti-HNA-1a IgM antibodies were detected in the serum of one female donor. Two of the other donors who contributed to the pooled platelet concentrate containing the HNA-1a IgM antibodies typed as HNA-1a/HNA-1b. CONCLUSION: Anti-HNA-1a IgM antibodies may have formed immune complexes with white cell fragments or soluble FcgammaRIII from HNA-1a+ donors in the pooled platelet concentrate and initiated TRALI.

Fetal and embryonic hemoglobins in erythroblasts of chromosomally normal and abnormal fetuses at 10-40 weeks of gestation.

BACKGROUND AND OBJECTIVES: During fetal development a change in erythropoiesis from hepatic to medullary site occurs. In chromosomally abnormal fetuses this change is delayed. Hemoglobin production also undergoes developmental switches from embryonic to fetal hemoglobins in the first trimester of pregnancy. The aim of study was to determine the proportion of embryonic and fetal hemoglobins in fetal erythroblasts of chromosomally normal and abnormal fetuses at 10-40 weeks of gestation. DESIGN AND METHODS: Fetal blood was obtained from 93 chromosomally normal and 19 abnormal fetuses at 10-40 weeks of gestation. Fetal erythroblasts were isolated by triple density gradient centrifugation and magnetic cell sorting with CD71 antibody. Fluorescent antibodies were used to immuno-stain for zeta (zeta), epsilon (epsilon) and gamma (gamma) hemoglobin chains. RESULTS: The percentages of the positively stained cells were calculated. In chromosomally normal fetuses the percentage of erythroblasts expressing the zeta chain was 25% at 10 weeks but this decreased exponentially with gestation to less than 1% by 17 weeks. Similarly, the percentage of cells expressing the epsilon chain decreased from 97% at 10 weeks to less than 1% by 25 weeks. In contrast, expression of the gamma chain increased from about 30% at 10 weeks to 90% by 16 weeks and decreased thereafter to 60% at 40 weeks. In the abnormal fetuses, the percentage of erythroblasts expressing the zeta chain and the epsilon chain decreased to less than 1% by 23 and 28 weeks respectively, while maximum expression of the gamma chain was at about 22 weeks. INTERPRETATION AND CONCLUSIONS: In the chromosomally abnormal group the pattern of change in the expression of the various hemoglobin chains during gestation was similar to that in the normal fetuses but was delayed by three to six weeks. These findings suggest that in fetuses with chromosomal abnormalities there is a developmental delay in the switch from embryonic to fetal hemoglobin chains.

Increased fetal erythroblasts in women who subsequently develop pre-eclampsia.

In pregnancies complicated by pre-eclampsia (PET) and/or intrauterine growth restriction (IUGR) there is an increased number of fetal cells in the maternal circulation. The aim of this study was to investigate whether this increase in fetal cells precedes the onset of these pregnancy complications. Doppler ultrasound studies at 24 weeks gestation have shown that increased impedance to flow in the uterine arteries identifies pregnancies with impaired placental perfusion that subsequently develop PET and/or IUGR. We obtained maternal blood from 18 pregnancies with abnormal Doppler results at 22-24 weeks gestation and from 10 normal controls. Fetal erythroblasts were enriched from maternal blood by triple density gradient centrifugation and magnetic cell sorting with CD71 antibody, and the percentage of these erythroblasts was determined. The median proportion of fetal erythroblasts in the group with abnormal Doppler results was 4.5% (range 1-12%), which was significantly higher than in the control group [median 1% (range 0-3%; P < 0.001)]. Furthermore, within the group with abnormal Doppler the median proportion of fetal erythroblasts was higher in the 10 cases which subsequently developed PET and/or IUGR [median 5.5% (range 3-12%)], than in those with normal pregnancy outcome [median 2% (range 1-5%; P < 0.01)]. These findings suggest that impaired placental perfusion is associated with an increase in feto-maternal cell traffic, which precedes the onset of PET and/or IUGR by several weeks.

Fetal cells in maternal blood of pregnancies with severe fetal growth restriction.

The aim of this study was to examine whether, in pregnancies with severe early onset fetal growth restriction, the number of fetal erythroblasts in maternal blood is increased. The percentage of fetal erythroblasts in maternal blood, enriched by triple density gradient centrifugation and anti-CD71 magnetic cell sorting, was determined in 10 singleton pregnancies with severe intrauterine growth restriction in which there was Doppler ultrasound evidence of impaired placental perfusion. The values were compared to those of 10 normal pregnancies at the same gestational range of 22-26 weeks. In the growth restricted pregnancies the median number of fetal erythroblasts per 100 nucleated cells in maternal blood enriched for fetal cells was significantly higher than the median value in the control pregnancies (8.5% compared with 1%; P < 0.001). These data suggest that impaired uteroplacental perfusion and severe fetal growth restriction may be associated with placental damage leading to increased feto-maternal cell traffic. Alternatively the rate of transfer of fetal cells into the maternal circulation is not altered but in growth restriction the proportion of fetal erythroblasts in fetal blood is increased.

Erythropoietin depletion and anaemia in diabetes mellitus.

AIMS: To discover whether Type 1 diabetic patients with autonomic neuropathy might be anaemic and erythropoietin (EPO)-depleted. METHODS: Fifteen Type 1 diabetic patients with serious complications (DM-COMP) were selected because of severe symptomatic autonomic neuropathy, including significant postural hypotension. All had proteinuria from nephropathy (three microalbuminuria and 12 macroalbuminuria), but a normal serum creatinine (< 122 micromol/l). They were compared to age and duration matched Type 1 diabetic controls without autonomic neuropathy (DM-controls) and non-diabetic patients with and without hypochromic, microcytic anaemia. RESULTS: The DM-COMP patients were anaemic (mean haemoglobin (Hb) 11.1+/-1.2 g/dl), sometimes severely (minimum Hb 9.2 g/dl), compared to non-neuropathic DM-controls (Hb 13.7+/-0.7 g/dl; P < 0.001). Furthermore, EPO failed to increase in association with anaemia in the DM-COMP group compared to the progressive increase in the non-diabetic, anaemic patients (difference of regression lines P < 0.001), indicating EPO depletion in the anaemic, diabetic patients. There was no other demonstrable cause for the anaemia. Treatment with EPO in 5 DM-COMP patients led to a rapid increase in haemoglobin (range 1.7-5.0 g/dl) with improvement in wellbeing. CONCLUSION: Some Type 1 diabetic patients with autonomic neuropathy present with an EPO-depleted anaemia, which responds to treatment with EPO. This observation supports the concept of autonomic neuropathy as a cause of anaemia with EPO depletion, although the role of established renal damage cannot be excluded.

Growth of Viridans streptococci on human serum alpha1-acid glycoprotein.

Viridans streptococci have emerged as major opportunistic pathogens. We suggest that for these bacteria to proliferate in vivo and cause disease, they must utilize host tissue components. We have therefore examined the ability of all recognized species of viridans streptococci to liberate and utilize the constituent sugars of the glycans of the extensively sialylated human serum alpha1-acid glycoprotein (AGP) as the sole source of carbohydrate to support in vitro growth. Analysis of residual glycans following bacterial growth was performed by high-pH anion exchange chromatography with pulsed amperometric detection and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Only those species which produced sialidase-namely, Streptococcus oralis, S. intermedius, and S. defectivus--grew on AGP. The extent of degradation of glycans was dependent on the particular glycosidases produced by the bacteria. S. defectivus produced only a sialidase which released the terminal N-acetylneuraminic acid residues of the glycans, and the liberated sugar was utilized. S. intermedius also produced beta-galactosidase and beta-N-acetylglucosaminidase, which removed galactose and N-acetylglucosamine from desialylated glycans, all of which again were utilized by the organism. S. oralis produced beta-galactosidase, beta-N-acetylglucosaminidase, and alpha-fucosidase and novel alpha- and beta-mannosidases which were apparent only from the analysis of the residual sugars of AGP. S. oralis cleaved all the sugars from AGP except for 22% of the N-acetylglucosamine. The residual N-acetylglucosamine residues remaining were those linked to the asparagine of the peptide backbone. All the monosaccharides released by S. oralis from AGP, with the exception of fucose, were utilized. Sialidase production may be a key factor for growth of these species of viridans streptococci on glycoproteins in vivo, since they are commonly associated with extra-oral diseases, with S. oralis emerging as an important pathogen.

Investigation of maternal blood enriched for fetal cells: role in screening and diagnosis of fetal trisomies.

Prenatal diagnosis of chromosomal abnormalities relies on assessment of risk followed by invasive testing in the group with highest risk. Assessment of risk by a combination of maternal age and fetal nuchal translucency and invasive testing in the 5% of the population with the highest risk would identify about 80% of trisomy 21 pregnancies. Preliminary reports suggest that chromosomal abnormalities can also be diagnosed by fluorescent in situ hybridization (FISH) in maternal blood enriched for fetal cells. This study examines the potential role of this method on the prenatal diagnosis of fetal trisomies. Maternal blood was obtained before invasive testing in 230 pregnancies at 10-14 weeks of gestation. After enrichment for fetal cells, by triple density centrifugation and anti-CD71 magnetic cell sorting, FISH was performed and the proportion of cells with positive signals in the chromosomally normal and abnormal groups was determined. Fetal karyotype was normal in 150 cases and abnormal in 80 cases, including 36 with trisomy 21. Using a 21 chromosome-specific probe, three-signal nuclei were present in at least 5% of the enriched cells from 61% of the trisomy 21 pregnancies and in none of the normal pregnancies. For a cut-off of 3% of three-signal nuclei the sensitivity for trisomy 21 was 97% for a false positive rate of 13%. Similar values were obtained in trisomies 18 and 13 using the appropriate chromosome-specific probe. Examination of fetal cells from maternal blood may provide a noninvasive prenatal diagnostic test for trisomy 21 with the potential of identifying about 60% of affected pregnancies. Alternatively, this technique can be combined with maternal age and fetal nuchal translucency as a method of selecting the high-risk group for invasive testing. Potentially, 80% of trisomy 21 pregnancies could be identified after invasive testing in less than 1% of the pregnant population.

Audit of practice in platelet refractoriness.

OBJECTIVES: A significant number of patients become refractory to platelet transfusion and prompt investigation of the cause will encourage appropriate selection of platelet products. METHODS: We surveyed haematologists to assess perceived practice concerning platelet refractoriness because of the high cost and limited availability of HLA-compatible platelets. Some 56 of 58 consultant haematologists participated. RESULTS: Clinicians differed on their definition of platelet refractoriness, and non-immune factors were not considered as important as immune causes of platelet refractoriness. A working group, including an invited moderator, was established to produce guidelines on recommended practice for the management of platelet refractoriness. Re-audit after implementation of the guidelines showed that more patients receiving HLA-compatible platelets had been tested for HLA antibodies. There was a mean 50.9% reduction in the use of HLA-compatible platelets. CONCLUSIONS: Increased testing for leucocyte and platelet antibodies resulted in reduced demand for and more selective use of HLA-compatible platelets, with no apparent increase in haemorrhagic complications.