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Maternal plasma fetal DNA as a marker for preterm labour
RESEARCH LETTERS
Low-molecular-weight heparin for immediate management of thromboembolic disease in pregnancy Andrew J Thomson, Isobel D Walker, Ian A Greer
Confidential Enquires into Maternal Deaths have highlighted the need for adequate treatment of thromboembolic disease, a major cause of maternal death in the UK.1 Low-molecular-weight heparin (LMWH) is effective, safe, and associated with fewer side-effects than unfractionated heparin for the treatment of acute deep vein thrombosis and pulmonary thromboembolism in nonpregnant women.2,3 LMWHs are commonly used for thromboprophylaxis during pregnancy,3 but unfractionated heparin is the mainstay of treatment for antenatal venous thromboembolism. The monitoring of unfractionated heparin by activated partial thromboplastin time can, however, be difficult in late pregnancy, when an apparent heparin resistance occurs because of increased fibrinogen and factor VIII.4,5 To date there has been no report of LMWH for treatment of deep venous thrombosis or pulmonary thromboembolism in pregnancy. We treated with subcutaneous enoxaparin, a LMWH, two women with antenatal deep vein thrombosis and two with antenatal pulmonary thromboembolism, objectively confirmed by ultrasound or ventilation-perfusion lung scintigraphy. The starting dose was 1 mg/kg every 12 h, based on the early pregnancy weight, since LMWH does not cross the placenta. We measured anti-Xa activity by chromogenic substrate assay (target therapeutic range 0·4–1·0 U/mL 3 h after injection). In one woman (weight 94 kg), the start dose was decreased from 100 mg to 80 mg twice daily since the peak concentration of anti-Xa on 100 mg was 1·2 U/mL; the resultant peak concentration of antiXa on 80 mg was 1·0 U/mL. In the other three women, the start dose of enoxaparin produced satisfactory peak concentrations of anti-Xa. We measured blood platelets, activated partial thromboplastin times, and anti-Xa concentrations before and weekly during treatment, and all of the women were continued on enoxaparin until delivery, (times between thromboembolism and delivery 1, 2, 4, and 8 weeks). The women were taught to self-inject, and three were managed as outpatients until delivery at term. The dose of enoxaparin was decreased to 40 mg every 12 h during labour, and recommenced immediately after delivery. Two
Anti-X a activity (U/mL)
0·8
1
2
3
4
5
Department of Health, Welsh Office, Scottish Home, and Health Department and Department of Health and Social Services Northern Ireland. Confidential Enquiries into Maternal Deaths in the United Kingdom 1994–96. London: Stationery Office, 1998. Simmoneau G, Sors H, Charbonnier B, et al. A comparison of lowmolecular weight heparin with unfractionated heparin for acute pulmonary embolism. N Engl J Med 1997; 337: 663–69. Nelson-Piercy C. Hazards of heparin. In: Greer IA, ed. Thromboembolic disease in obstetrics and gynaecology. Balliéte’s Clin Obstet Gynaecol 1997; 11: 489–509. Greer IA. Special case of venous thromboembolism in pregnancy. In Tooke JE, Lowe GDO, eds. A textbook of vascular medicine. London: Arnold, 1996. Hyers TM, Hull RD, Weg JG. Antithrombotic therapy for venous thromboembolic disease. Chest 1995; 108 (suppl): 335S–51.
Departments of Obstetrics and Haematology, Glasgow Royal Maternity Hospital and Glasgow Royal Infirmary, Glasgow, G31 2ER, UK (I A Greer)
Maternal plasma fetal DNA as a marker for preterm labour Tse N Leung, Jun Zhang, Tze K Lau, N Magnus Hjelm, Y M Dennis Lo
0·6
0·4
0·2
0 0
10
20
30
40 50 60 Time (h)
70
80
Anti-Xa activity in a patient with acute pulmonary thromboembolism on 60 mg enoxaparin twice daily
1904
women had epidural anaesthesia without complication. The fourth woman stayed in hospital and was delivered under spinal anaesthetic by caesarean section for placenta praevia. Intrapartum blood loss for each woman was within normal limits. Median peak (3 h) anti-Xa activity in the four women was 0·67 U/mL (range 0·46–1·00 on 18 observations). In one woman (weight 58 kg), a 24 h anti-Xa activity profile showed that a measurable protective anticoagulant effect (activity >0·2 U/mL) was maintained between drug administration (figure). Furthermore, in each woman, there was no cumulative anticoagulant effect. One woman, treated for 8 weeks, had monthly peak concentrations of anti-Xa of 1·00 U/mL, 1·00 U/mL, and 0·89 U/mL. The activated partial thromboplastin time for each woman remained within normal during enoxaparin treatment and no woman developed thrombocytopenia. Subcutaneous LMWH seems to have advantages over unfractionated heparin for venous thromboembolism in pregnancy. The simplified therapeutic regimen for LMWH enables patients to be treated as outpatients. LMWHs have a higher anti-factor Xa activity/anti-factor IIa activity ratio than unfractionated heparin and a more predictable dose response. LMWH may be a safe and effective treatment for venous thromboembolism in pregnancy with easy administration. Assessment of anti-Xa during treatment should, however, be carried out until greater experience is obtained.
90 100
The discovery of high concentrations of fetal DNA in maternal plasma in the last 8 weeks of pregnancy1 has led to new opportunities for clinical application.2 Although the biological basis of this rise in concentration remains unclear, one hypothesis suggests that it is due to the breaking down of the placental barrier in anticipation of delivery.3 If this hypothesis is true, we propose that raised fetal DNA concentrations will be seen in maternal plasma before spontaneous preterm delivery. We recruited pregnant women admitted with suspected preterm labour at or before 34 weeks’ gestation, with informed consent, from the obstetrics department of the Chinese University of Hong Kong. Gestational age was confirmed by ultrasound examination. The Clinical Research Ethics Committee approved the study. The diagnosis of preterm labour was made according to the
THE LANCET • Vol 352 • December 12, 1998
RESEARCH LETTERS
Fetal DNA concentration (copies/mL)
1000
800
delivery. Measurement of fetal DNA might be useful as a marker of preterm delivery, which is the single most important cause of perinatal morbidity and mortality. Since lower concentrations of fetal DNA were associated with successful tocolytic therapy, this technique may help to differentiate true and false preterm labour.
Control Preterm delivery, failed tocolysis Preterm delivery, no tocolysis Threatened preterm labour, successful tocolysis
1
600 2
400
3 4
200
Lo YMD, Tein MSC, Lau TK, et al. Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis. Am J Hum Genet 1998; 62: 768–75. Lo YMD, Corbetta N, Chamberlain PF, et al. Presence of fetal DNA in maternal plasma and serum. Lancet 1997; 350: 485–87. Bianchi DW. Fetal DNA in maternal plasma: the plot thickens and the placental barrier thins. Am J Hum Genet 1998; 62: 763–64. The Canadian preterm labour investigators group. Treatment of preterm labour with the beta-adrenergic agonist ritodrine. N Engl J Med 1992; 327: 308–12.
Departments of Obstetrics and Gynaecology and Chemical Pathology (Y M D Lo), The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR
0 Control
Preterm delivery
Suspected preterm labour
Concentrations of fetal DNA by group
definition of the Canadian preterm labour investigations group.4 We took 10 mL samples of venous blood on admission from maternal antecubital veins into heparinised tubes for the isolation of plasma. We used 400–800 L of plasma for DNA extraction with a QIAamp Blood Kit (Qiagen, Hilden, Germany).1 The SRY gene on the Y chromosome was used as a marker for male fetuses and fetal SRY sequence was detected with real-time quantitative PCR.1 20 women had spontaneous preterm deliveries at between 26 weeks’ and 34 weeks’ gestation (median 31·5 weeks). Nine women delivered on the day of blood sampling. The longest time between blood sampling and delivery was 8 days. Control blood samples were collected from the same number of healthy pregnant women admitted to the same unit during the period of the study and matched for gestational age (median 31·2 weeks). All controls had term deliveries. Fetal-derived SRY signal was detected in 13 women with preterm delivery and 17 controls who had male fetuses. Tocolytic therapy was tried but not effective in eight women in the preterm group who had male fetuses, and was not tried in remaining five women with male fetuses. No SRY signal was found in the women in the two groups who had female fetuses. The median fetal DNA concentrations in preterm and control pregnancies were 124·8 copies/mL (IQR 67·8–568·2) and 65·8 copies/mL (44·3–143·3), respectively. Fetal DNA concentrations were significantly higher in preterm than control pregnancies (Mann-Whitney U test; p=0·042; figure). We also investigated whether fetal DNA measurement enabled differentiation between true and false preterm labour. 12 women were recruited who presented with threatened preterm labour between 24 weeks’ and 33 weeks’ gestation (median 30·3 weeks) in whom labour was successfully suppressed by tocolysis, leading to term delivery. SRY signals were obtained from the six women carrying male fetuses. The median fetal DNA concentration was 46·3 copies/mL (8·8–79·4), which was significantly lower than that of the eight women in the preterm group carrying male fetuses in whom tocolytic treatment was not effective (median 119·3 copies/mL [78·8–439·1]; p=0·017; figure). There was no significant difference between women successfully treated by tocolytic therapy in the two groups (p=0·23). High concentrations of fetal DNA were, therefore, detectable in maternal plasma before spontaneous preterm
THE LANCET • Vol 352 • December 12, 1998
Expression of CD38 on CD8 T cells predicts maintenance of high viraemia in HAART-treated HIV-1-infected children Alessandra Viganó, Marina Saresella, Stefano Rusconi, Pasquale Ferrante, Mario Clerici
Highly active antiretroviral therapy (HAART) is capable of suppressing viral replication, reducing plasma HIV-1-viral load, and increasing CD4 T cells in the majority of HIV-1infected people. Inhibition of HIV-1 replication is not achieved in all people treated.1 No immune or virological correlate markers have a predictive value for the lack of virological effect of HAART. We report that the presence of an augmented percentage of CD38 expressing CD8 (CD8 CD38) T lymphocytes is associated with the persistence of high plasma viraemia in HAART-treated children and that down-modulation of CD8 CD38 T lymphocytes is only seen in people in whom HAART-associated suppression of HIV-1 replication is detected. We followed 16 vertically infected children (mean age=8·2 years [95% CI 5·5–14·6]). The diagnosis of HIV-1 infection and the clinical and immunological status of the patients met the criteria of the 1994 revised Centers for Disease Control and Prevention (CDC) HIV-1 classification for children.2 All had moderate-to-advanced disease (CDC classification=5A2; 5B3; and 6C3); all had been treated with zidovudine alone (six patients) or associated with didanosine (nine patients) and were subsequently switched to HAART (lamivudine [4 mg/kg per 12 h]+stavudine [1 mg/kg per 12 h]+indinavir [500 mg/m2 per 8 h]). Virological and immunological tests were done before the start of treatment (time 0) and after 3, 6, 9, and 12 months. HAART induced a large reduction in HIV-1 plasma viraemia in 14/16 patients (time 0=median 27 000 SE 3230 RNA copies/mL; plasma viraemia <400 RNA copies/mL at all subsequent tests). HIV-1 viraemia was only slightly and temporarily reduced in the two remaining patients (RNA copies/mL: patient OG; time 0=490 000; 3=8000; 6=89 000; 9=150 000; 12 months=60 000; patient LL; time 0=82 000; 3=4000; 6=41 000; 9=37 000; 12 months=33 000); (figure). No correlation was detected between lack of response to HAART and age, disease stage, or previous treatment. Analysis of viral RNA in plasma for drug resistance was done in the two patients who remained viraemic; in both cases, multiple drug resistance-
1905
Low-molecular-weight heparin for immediate management of thromboembolic disease in pregnancy Andrew J Thomson, Isobel D Walker, Ian A Greer
Confidential Enquires into Maternal Deaths have highlighted the need for adequate treatment of thromboembolic disease, a major cause of maternal death in the UK.1 Low-molecular-weight heparin (LMWH) is effective, safe, and associated with fewer side-effects than unfractionated heparin for the treatment of acute deep vein thrombosis and pulmonary thromboembolism in nonpregnant women.2,3 LMWHs are commonly used for thromboprophylaxis during pregnancy,3 but unfractionated heparin is the mainstay of treatment for antenatal venous thromboembolism. The monitoring of unfractionated heparin by activated partial thromboplastin time can, however, be difficult in late pregnancy, when an apparent heparin resistance occurs because of increased fibrinogen and factor VIII.4,5 To date there has been no report of LMWH for treatment of deep venous thrombosis or pulmonary thromboembolism in pregnancy. We treated with subcutaneous enoxaparin, a LMWH, two women with antenatal deep vein thrombosis and two with antenatal pulmonary thromboembolism, objectively confirmed by ultrasound or ventilation-perfusion lung scintigraphy. The starting dose was 1 mg/kg every 12 h, based on the early pregnancy weight, since LMWH does not cross the placenta. We measured anti-Xa activity by chromogenic substrate assay (target therapeutic range 0·4–1·0 U/mL 3 h after injection). In one woman (weight 94 kg), the start dose was decreased from 100 mg to 80 mg twice daily since the peak concentration of anti-Xa on 100 mg was 1·2 U/mL; the resultant peak concentration of antiXa on 80 mg was 1·0 U/mL. In the other three women, the start dose of enoxaparin produced satisfactory peak concentrations of anti-Xa. We measured blood platelets, activated partial thromboplastin times, and anti-Xa concentrations before and weekly during treatment, and all of the women were continued on enoxaparin until delivery, (times between thromboembolism and delivery 1, 2, 4, and 8 weeks). The women were taught to self-inject, and three were managed as outpatients until delivery at term. The dose of enoxaparin was decreased to 40 mg every 12 h during labour, and recommenced immediately after delivery. Two
Anti-X a activity (U/mL)
0·8
1
2
3
4
5
Department of Health, Welsh Office, Scottish Home, and Health Department and Department of Health and Social Services Northern Ireland. Confidential Enquiries into Maternal Deaths in the United Kingdom 1994–96. London: Stationery Office, 1998. Simmoneau G, Sors H, Charbonnier B, et al. A comparison of lowmolecular weight heparin with unfractionated heparin for acute pulmonary embolism. N Engl J Med 1997; 337: 663–69. Nelson-Piercy C. Hazards of heparin. In: Greer IA, ed. Thromboembolic disease in obstetrics and gynaecology. Balliéte’s Clin Obstet Gynaecol 1997; 11: 489–509. Greer IA. Special case of venous thromboembolism in pregnancy. In Tooke JE, Lowe GDO, eds. A textbook of vascular medicine. London: Arnold, 1996. Hyers TM, Hull RD, Weg JG. Antithrombotic therapy for venous thromboembolic disease. Chest 1995; 108 (suppl): 335S–51.
Departments of Obstetrics and Haematology, Glasgow Royal Maternity Hospital and Glasgow Royal Infirmary, Glasgow, G31 2ER, UK (I A Greer)
Maternal plasma fetal DNA as a marker for preterm labour Tse N Leung, Jun Zhang, Tze K Lau, N Magnus Hjelm, Y M Dennis Lo
0·6
0·4
0·2
0 0
10
20
30
40 50 60 Time (h)
70
80
Anti-Xa activity in a patient with acute pulmonary thromboembolism on 60 mg enoxaparin twice daily
1904
women had epidural anaesthesia without complication. The fourth woman stayed in hospital and was delivered under spinal anaesthetic by caesarean section for placenta praevia. Intrapartum blood loss for each woman was within normal limits. Median peak (3 h) anti-Xa activity in the four women was 0·67 U/mL (range 0·46–1·00 on 18 observations). In one woman (weight 58 kg), a 24 h anti-Xa activity profile showed that a measurable protective anticoagulant effect (activity >0·2 U/mL) was maintained between drug administration (figure). Furthermore, in each woman, there was no cumulative anticoagulant effect. One woman, treated for 8 weeks, had monthly peak concentrations of anti-Xa of 1·00 U/mL, 1·00 U/mL, and 0·89 U/mL. The activated partial thromboplastin time for each woman remained within normal during enoxaparin treatment and no woman developed thrombocytopenia. Subcutaneous LMWH seems to have advantages over unfractionated heparin for venous thromboembolism in pregnancy. The simplified therapeutic regimen for LMWH enables patients to be treated as outpatients. LMWHs have a higher anti-factor Xa activity/anti-factor IIa activity ratio than unfractionated heparin and a more predictable dose response. LMWH may be a safe and effective treatment for venous thromboembolism in pregnancy with easy administration. Assessment of anti-Xa during treatment should, however, be carried out until greater experience is obtained.
90 100
The discovery of high concentrations of fetal DNA in maternal plasma in the last 8 weeks of pregnancy1 has led to new opportunities for clinical application.2 Although the biological basis of this rise in concentration remains unclear, one hypothesis suggests that it is due to the breaking down of the placental barrier in anticipation of delivery.3 If this hypothesis is true, we propose that raised fetal DNA concentrations will be seen in maternal plasma before spontaneous preterm delivery. We recruited pregnant women admitted with suspected preterm labour at or before 34 weeks’ gestation, with informed consent, from the obstetrics department of the Chinese University of Hong Kong. Gestational age was confirmed by ultrasound examination. The Clinical Research Ethics Committee approved the study. The diagnosis of preterm labour was made according to the
THE LANCET • Vol 352 • December 12, 1998
RESEARCH LETTERS
Fetal DNA concentration (copies/mL)
1000
800
delivery. Measurement of fetal DNA might be useful as a marker of preterm delivery, which is the single most important cause of perinatal morbidity and mortality. Since lower concentrations of fetal DNA were associated with successful tocolytic therapy, this technique may help to differentiate true and false preterm labour.
Control Preterm delivery, failed tocolysis Preterm delivery, no tocolysis Threatened preterm labour, successful tocolysis
1
600 2
400
3 4
200
Lo YMD, Tein MSC, Lau TK, et al. Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis. Am J Hum Genet 1998; 62: 768–75. Lo YMD, Corbetta N, Chamberlain PF, et al. Presence of fetal DNA in maternal plasma and serum. Lancet 1997; 350: 485–87. Bianchi DW. Fetal DNA in maternal plasma: the plot thickens and the placental barrier thins. Am J Hum Genet 1998; 62: 763–64. The Canadian preterm labour investigators group. Treatment of preterm labour with the beta-adrenergic agonist ritodrine. N Engl J Med 1992; 327: 308–12.
Departments of Obstetrics and Gynaecology and Chemical Pathology (Y M D Lo), The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR
0 Control
Preterm delivery
Suspected preterm labour
Concentrations of fetal DNA by group
definition of the Canadian preterm labour investigations group.4 We took 10 mL samples of venous blood on admission from maternal antecubital veins into heparinised tubes for the isolation of plasma. We used 400–800 L of plasma for DNA extraction with a QIAamp Blood Kit (Qiagen, Hilden, Germany).1 The SRY gene on the Y chromosome was used as a marker for male fetuses and fetal SRY sequence was detected with real-time quantitative PCR.1 20 women had spontaneous preterm deliveries at between 26 weeks’ and 34 weeks’ gestation (median 31·5 weeks). Nine women delivered on the day of blood sampling. The longest time between blood sampling and delivery was 8 days. Control blood samples were collected from the same number of healthy pregnant women admitted to the same unit during the period of the study and matched for gestational age (median 31·2 weeks). All controls had term deliveries. Fetal-derived SRY signal was detected in 13 women with preterm delivery and 17 controls who had male fetuses. Tocolytic therapy was tried but not effective in eight women in the preterm group who had male fetuses, and was not tried in remaining five women with male fetuses. No SRY signal was found in the women in the two groups who had female fetuses. The median fetal DNA concentrations in preterm and control pregnancies were 124·8 copies/mL (IQR 67·8–568·2) and 65·8 copies/mL (44·3–143·3), respectively. Fetal DNA concentrations were significantly higher in preterm than control pregnancies (Mann-Whitney U test; p=0·042; figure). We also investigated whether fetal DNA measurement enabled differentiation between true and false preterm labour. 12 women were recruited who presented with threatened preterm labour between 24 weeks’ and 33 weeks’ gestation (median 30·3 weeks) in whom labour was successfully suppressed by tocolysis, leading to term delivery. SRY signals were obtained from the six women carrying male fetuses. The median fetal DNA concentration was 46·3 copies/mL (8·8–79·4), which was significantly lower than that of the eight women in the preterm group carrying male fetuses in whom tocolytic treatment was not effective (median 119·3 copies/mL [78·8–439·1]; p=0·017; figure). There was no significant difference between women successfully treated by tocolytic therapy in the two groups (p=0·23). High concentrations of fetal DNA were, therefore, detectable in maternal plasma before spontaneous preterm
THE LANCET • Vol 352 • December 12, 1998
Expression of CD38 on CD8 T cells predicts maintenance of high viraemia in HAART-treated HIV-1-infected children Alessandra Viganó, Marina Saresella, Stefano Rusconi, Pasquale Ferrante, Mario Clerici
Highly active antiretroviral therapy (HAART) is capable of suppressing viral replication, reducing plasma HIV-1-viral load, and increasing CD4 T cells in the majority of HIV-1infected people. Inhibition of HIV-1 replication is not achieved in all people treated.1 No immune or virological correlate markers have a predictive value for the lack of virological effect of HAART. We report that the presence of an augmented percentage of CD38 expressing CD8 (CD8 CD38) T lymphocytes is associated with the persistence of high plasma viraemia in HAART-treated children and that down-modulation of CD8 CD38 T lymphocytes is only seen in people in whom HAART-associated suppression of HIV-1 replication is detected. We followed 16 vertically infected children (mean age=8·2 years [95% CI 5·5–14·6]). The diagnosis of HIV-1 infection and the clinical and immunological status of the patients met the criteria of the 1994 revised Centers for Disease Control and Prevention (CDC) HIV-1 classification for children.2 All had moderate-to-advanced disease (CDC classification=5A2; 5B3; and 6C3); all had been treated with zidovudine alone (six patients) or associated with didanosine (nine patients) and were subsequently switched to HAART (lamivudine [4 mg/kg per 12 h]+stavudine [1 mg/kg per 12 h]+indinavir [500 mg/m2 per 8 h]). Virological and immunological tests were done before the start of treatment (time 0) and after 3, 6, 9, and 12 months. HAART induced a large reduction in HIV-1 plasma viraemia in 14/16 patients (time 0=median 27 000 SE 3230 RNA copies/mL; plasma viraemia <400 RNA copies/mL at all subsequent tests). HIV-1 viraemia was only slightly and temporarily reduced in the two remaining patients (RNA copies/mL: patient OG; time 0=490 000; 3=8000; 6=89 000; 9=150 000; 12 months=60 000; patient LL; time 0=82 000; 3=4000; 6=41 000; 9=37 000; 12 months=33 000); (figure). No correlation was detected between lack of response to HAART and age, disease stage, or previous treatment. Analysis of viral RNA in plasma for drug resistance was done in the two patients who remained viraemic; in both cases, multiple drug resistance-
1905