http://informahealthcare.com/jmf ISSN: 1476-7058 (print), 1476-4954 (electronic) J Matern Fetal Neonatal Med, Early Online: 1–5 ! 2014 Informa UK Ltd. DOI: 10.3109/14767058.2014.940891
ORIGINAL ARTICLE
Establishing reference intervals for D-dimer to trimesters S¸ erif Ercan1, Sadullah O¨zkan2, Nihal Yu¨cel3, and Asuman Orc¸un3 1
Department of Clinical Biochemistry, Lu¨leburgaz State Hospital, Kırklareli, Turkey, 2Department of Obstetric and Gynecology, and Department of Clinical Biochemistry, Dr. Lutfi Kirdar Kartal Education and Research Hospital, Istanbul, Turkey
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Abstract
Keywords
Objective: When D-dimer is used to evaluate suspected venous thromboembolism in pregnant patients, the reference interval of common population may cause misinterpretation. The present study aims to determine reference intervals of D-dimer in the three trimesters. Methods: Four-hundred sixteen pregnant women and 32 non-pregnant women were enrolled in this cross-sectional study. Reference group had comprised 123 pregnant in the first trimester (5–11 week), 164 pregnant in the second trimester (13–20 week) and 126 pregnant women in the third trimester (25–35 week). D-dimer levels were analyzed via immunoturbidimetric assay. Results: If the threshold of 0.50 mg/L for diagnosis of VTE is used, 4.8% of pregnant women in the second trimester and 23.8% of pregnant women in the third trimester would have D-dimer levels exceeding this cut-off value. Reference intervals of D-dimer were determined as 0.11–0.40 mg/L; 0.14–0.75 mg/L and 0.16–1.3 mg/L in first, second and third trimester, respectively. Conclusion: The established D-dimer reference intervals for each trimester of pregnancy are different from those used in common population. These reference intervals may assist clinicians in making accurate clinical decisions. Further studies are needed to establish new cut-off values for the D-dimer to rule out VTE in each trimester.
D-dimer, pregnancy, reference interval, trimesters, venous thromboembolism
Introduction Normal pregnancy is a hypercoagulable state because of increased concentration of procoagulants, decreased levels of anticoagulants and diminished fibrinolytic activity [1]. The hypercoagulable state protects pregnant women from the bleeding complications of miscarriage and childbirth. However, the risk of venous thromboembolism (VTE) is increased 4- to 5-fold in pregnant women compared to women who are not pregnant. The overall prevalence of thromboembolic events during pregnancy is approximately 2 per 1000 deliveries. VTE accounts for 1.1 deaths per 100 000 deliveries, or 10% of all maternal deaths [2]. Diagnosis of VTE in pregnancy is challenging due to many of the classic symptoms such as leg swelling and respiratory symptoms which are also associated with normal pregnancy [3]. In diagnostic imaging of deep venous thrombosis (DVT), contrast venography or compression ultrasonography has important limitations. Venography is invasive, expensive and exposes the patient to the radiation. Although compression ultrasound has high diagnostic sensitivity and specificity for
Address for correspondence: S ¸ erif Ercan, Department of Clinical ¨ zeri Yeni Biochemistry, Lu¨leburgaz State Hospital, D 100 Karayolu U Sanayi Kar¸s ısı 39750 Lu¨leburgaz/Kırklareli, Istanbul, Turkey. Tel: +90 5555875287. Fax: +90 2884131046. E-mail: serifercan@ yahoo.com.tr
History Received 4 July 2013 Revised 15 June 2014 Accepted 30 June 2014 Published online 25 July 2014
DVT without ionizing radiation, it is not always available in many clinics and small hospitals. In addition, it has poor sensitivity for detection of calf vein thrombosis, and sequential testing is often needed when the initial scan is negative. In the investigation of suspected pulmonary emboli (PE), all available imaging techniques such as scintillation ventilation-perfusion lung scan or a computed tomography angiography cause exposure of both the mother and fetus to the effects of ionizing radiation [3,4]. To diagnose the VTE in pregnancy; objective, rapid, inexpensive, non-invasive and harmless fetus tests are required. D-dimer, a degradation product of a cross-linked fibrin blood clot, has the potential to detect thrombosis in any part of the venous system. A negative value of D-dimer may safely rule out both DVT and PE with a high sensitivity up to 95% and a negative predictive value of almost 100% [5]. D-dimer is sensitive but non-specific marker for VTE because it is elevated in variety of non-thrombotic conditions such as recent major surgery, hemorrhage, trauma, cancer and pregnancy [6]. D-dimer levels increase progressively from trimester to trimester during normal pregnancy [7–10]. When the D-dimer cut-off value for common population is used to evaluate VTE in pregnant women, this increase may cause misinterpretation. The D-dimer assay has been suggested as an initial test to rule out VTE and reduce the number of patients requiring diagnostic imaging. However, D-Dimer role in algorithms for
2
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diagnosis of VTE in pregnancy has not been adequately studied. Moreover, D-dimer reference values derived from sufficient number of healthy pregnant women for different trimesters are not available. Therefore, prior to clinical application, we aimed in this study, to establish D-Dimer reference values for each trimester.
J Matern Fetal Neonatal Med Downloaded from informahealthcare.com by Columbia University on 03/01/15 For personal use only.
Methods This cross-sectional study included 448 healthy women including 416 pregnant and 32 non-pregnant women. Pregnant women followed by Dr. Lutfi Kirdar Kartal Training and Research Hospital, Department of Obstetrics and Gynecology were enrolled in the study. According to recommendation of International Federation of Clinical Chemistry and Clinical and Laboratory Standards Institute [11], healthy pregnant women for reference population were selected. The exclusion criteria were designed to minimize any possible influence on D-dimer levels: any personal history of thromboembolic disease, bleeding disorders, liver disease (abnormal liver function tests), renal disease (abnormal creatinine), autoimmune diseases such as systemic lupus erythematosus, pancreatitis (abnormal amylase concentrations with abdominal pain), morbid obesity (body mass index 40 kg/m2), a condition requiring daily injections (such as insulin-dependent diabetes mellitus) and current infection with fever 438 C. The additional exclusion criteria were defined as diastolic blood pressure 485 mmHg and proteinuria. This study has been carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans. The pregnant women were divided into three groups based on gestational age: first trimester (5–11 weeks, n ¼ 123), second trimester (13–20 weeks, n ¼ 166), and third trimester (25–35 weeks, n ¼ 127). Gestational age was determined by ultrasonographic biometric examination and the date of last menstrual period. When discrepancy between ultrasound and last menstrual period was higher than a week, gestational age was determined according to biometric values. A control group comprised age-matched healthy nonpregnant women were selected according to exclusion criteria used in pregnant group. Blood samples were obtained in follicular phase of menstruation cycle. Blood samples were drawn into a tube containing 0.11 mmol/L (3.2%) sodium citrate (one part anticoagulant plus nine parts blood). Plasma was prepared by centrifugation at 2000g for 10-min. D-dimer levels were measured in fresh, unfrozen plasma samples. Prothrombin time (PT), activated partial thromboplastin time (aPTT) and fibrinogen levels were also analyzed in these plasma samples. Analyses were performed within 4 hours of blood sampling. D-Dimer analysis was performed using the AMAX AUTO D-Dimer kit (Trinity Biotech Plc, Co Wicklow, Ireland) on the AMAX 400 automated coagulation analyzer (Trinity Biotech Plc). This analysis is based on immunoturbidimetric method. By conventional mechanic coagulometric techniques on the same coagulometer, PT, aPTT and fibrinogen levels were analyzed using TriniCLOT PT HTF, TriniCLOT aPTT HS and MDA Fibriquik reagents (Trinity Biotech Plc, Co Wicklow, Ireland), respectively.
J Matern Fetal Neonatal Med, Early Online: 1–5
Statistical analyses were performed using Statistical Package for Social Sciences (SPSS 15.0, SPSS Inc., Chicago, IL) in computerized media. Variables are presented as means ± SD and median according to data distribution. The significance of the mean differences was analyzed with Kruskal–Wallis test. If the Kruskal–Wallis test revealed significant differences, pair-wise comparisons with MannWhitney Test were used to identify the differences. p Values less than or equal to 0.05 were considered to be statistically significant. The MedCalc program (version 12.2.1.0) was used to calculate the non-parametric 2.5–97.5 percentile reference intervals for D-dimer. Reed method was used to determine outliers of data sets.
Results In this study, 416 pregnant women and 32 non-pregnant women were included. Two pregnant women were excluded from study because of abnormal PT results in the first and the second trimester groups. One pregnant woman was also excluded because of elevated aPTT result in the second trimester group. Reference population had finally comprised 123 pregnant in the first trimester (5–11 week), 164 pregnant in the second trimester (13–20 week) and 126 pregnant women in the third trimester (25–35 week). Median age of pregnant women was 27 years (ranging from 16 to 45 years), and that of non-pregnant women was 25 years (ranging from 17 to 35 years). There was no significant difference. The mean ± SD or median values of each parameter measured in pregnant and non-pregnant women are shown in Table 1. As compared to non-pregnant women, D-dimer and fibrinogen levels were found to be significantly elevated in each trimester (Figures 1 and 2, respectively; Table 1). In addition, aPTT was significantly shortened (p50.01) and PT was not changed (Table 1). D-dimer levels had significantly higher in the second trimester than those of the first trimester (median: 0.29 versus 0.18 mg/L, p50.001). D-dimer levels in the third trimester were also found to be significantly increased compared to the second trimester (median: 0.31 versus 0.29 mg/L, p50.001; Table 1). There was no significant difference of fibrinogen levels between the first and the second trimesters. However, fibrinogen levels in the third trimester were higher than those of the second trimester (median: 3.7 versus 3.4 g/L, p50.01; Table 1). There was no significant difference of PT values between the first and the second trimesters and between the second and the third trimesters. aPTT values in the second trimester were found to be elevated compared to the first trimester (p50.001), however there was no significant difference of aPTT values between the second and the third trimesters (Table 1). Reference intervals for PT and aPTT are 11.1–13.2 s and 22.6–35 s, respectively. PT and aPTT values were found to be within the reference intervals in almost all of the
D-dimer levels during pregnancy
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DOI: 10.3109/14767058.2014.940891
pregnant women. Two of the pregnant women had slightly elevated PT INR values. Only one pregnant woman had slightly elevated aPTT values. Twenty-three pregnant women in the first trimester, 48 pregnant women in the second trimester and 54 pregnant women in the third trimester had values that exceeded the upper limit of fibrinogen which was 3.8 g/L (Figure 2). In our laboratory, the upper limit for D-dimer used in common population was 0.15 mg/L. The control group fell below our current upper limit. Moreover, in accordance with the upper limit of common population, the upper limit for D-dimer for non-pregnant women was determined as 0.16 mg/L. D-dimer values of 117 of the pregnants in the first trimester, 162 of the pregnants in the second trimester and all the pregnants in the third trimester exceeded the upper limit of D-dimer. When the cut-off of 0.50 mg/L was used in diagnosis Table 1. The D-dimer, fibrinogen, PT and aPTT values in healthy pregnant and non-pregnant women.
Control n 32 D-dimer (mg/L) Median 0.12 Range 0.06–0.14 Fibrinogen (g/L) Median 3.1 Range 1.8–3.8 PT (s) Mean ±SD 11.9 ± 1.4 aPTT (s) Mean ± SD 34.2 ± 2.7
1st Trimester
2nd Trimester
3rd Trimester
123
164
126
0.18* 0.10–0.46
0.29*,** 0.10–1.46
0.31*,**,*** 0.13–1.75
3.5* 2.1–4.3
3.4* 1.5–7.1
3.7*,*** 2.2–5.9
12 ± 1.6
11.9 ± 1.6
11.8 ± 1.6
32.5 ± 3.4ô
30.5 ± 2.7ô,**
30.5 ± 2.8ô
*p50.001 versus control; **p50.001 versus 1st trimester; ***p50.001 versus 2nd trimester; ôp50.01 versus control.
Figure 1. Plasma D-dimer levels in healthy pregnant and non-pregnant women. Vertical point plot shows the D-dimer levels for each subject. The smooth horizontal bar indicates the cut-off value for VTE. Interrupted horizontal bar presents the upper limit of reference interval for non-pregnant women.
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of VTE, 8 the pregnant women in the second trimester and 30 pregnant women in the third trimester were found to exceed this threshold (Figure 1). By correlation analysis, we aimed to determine whether the increase in fibrinogen was correlated with the increase in D-dimer. A significant correlation was not identified between D-dimer and fibrinogen levels. The calculated reference intervals for D-dimer are presented in Table 2. The reference interval of D-dimer was established as 0.11–0.40, 0.14–0.75 and 0.16–1.3 mg/L in the first, second and third trimesters, respectively. For fibrinogen, we also calculated the reference interval as 2.5–4.3, 1.8–5.9 and 2.6–4.9 g/L in the first, second and third trimesters, respectively.
Discussion D-Dimer testing is quick, simple, and inexpensive and it has a high sensitivity of up to 95% and a negative predictive value of nearly 100% for VTE [5,12]. Therefore, D-dimer testing should be used to rule out VTE in order to reduce the number of patients requiring potentially harmful diagnostic imaging. Pregnant women have a two-to-five fold higher risk of VTE compared to non-pregnant women. The diagnosis of VTE during pregnancy is difficult due to physiologic changes and the possibility of differences in pathophysiology and presentation of VTE in pregnancy [3]. Although D-dimer testing has an important role in the exclusion of VTE in the non-pregnant population, D-Dimer incorporation in algorithms for diagnosing VTE in pregnant women has not been adequately studied. Furthermore, there is not adequate number of studies aimed to establish the reference values of D-dimer with a sufficient number of pregnant women for each trimester. Previous studies demonstrated that plasma D-dimer levels rise during pregnancy [7–10]. As expected, we found the
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J Matern Fetal Neonatal Med, Early Online: 1–5
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Figure 2. Plasma fibrinogen levels in healthy pregnant and non-pregnant women. Vertical point plot shows the fibrinogen levels for each subject. The horizontal bar indicates the upper limit of reference interval for non-pregnant women.
Table 2. The reference intervals of D-dimer and fibrinogen to trimesters and non-pregnant women.
D-Dimer (mg/L) Reference interval method 95% Reference intervals Fibrinogen (g/L) Reference interval method 95% Reference intervals
Controls
First trimester
Second trimester
Third trimester
Parametric 0.07–0.16
Non-parametric 0.11–0.40
Non-parametric 0.14–0.75
Non-parametric 0.16–1.3
Parametric 2.2–3.9
Parametric 2.5–4.3
Non-parametric 1.8–5.9
Parametric 2.6–4.9
increase of the D-dimer concentration during pregnancy from trimester to trimester. The highest D-dimer levels were obtained during the third trimester. We also determined that 95.1% of pregnant women in the first trimester, 98.7% of pregnant women in the second trimester, 100% of pregnant women in the third trimester had D-dimer levels that exceeded reference value which was up to 0.15 mg/L. If the threshold of 0.50 mg/L for diagnosis of VTE is used, 4.8% of pregnant women in the second trimester and 23.8% of pregnant women in the third trimester were found to exceed this cut-off value. The elevation of D-dimer levels from trimester to trimester restricts its use in the evaluation of suspected VTE in pregnant women. Determination of a cut-off value for VTE during pregnancy is difficult because it requires very expensive multicenter study which includes large numbers of pregnant women tested for VTE. Therefore, prior to clinical application, the determination of reference intervals of D-dimer for each trimester in a sufficient number of reference individuals is essential. International Federation of Clinical Chemistry and Clinical and Laboratory Standards Institute guidelines recommend establishing a reference interval in at least 120 reference individuals using the non-parametric ranking method [11]. Therefore, at least 120 pregnant women for each trimester were included into our study. By non-parametric method, we
determined the reference interval of D-dimer as: for the first trimester 0.11–0.40 mg/L; for the second trimester 0.14–0.75 mg/L and for the third trimester 0.16–1.3 mg/L. In a prospective study, Morse analyzed D-dimer levels throughout the pregnancy in 48 pregnant women [7]. Author calculated the reference interval of D-dimer as mean ± SD, which were 191 ± 25 ng/mL in 16 weeks, 393 ± 72 ng/mL in 26 weeks, 544 ± 96 ng/mL in 34 weeks and 140 ± 58 ng/mL in controls. Recently, Kovac et al. prospectively analyzed D-dimer levels in 89 healthy pregnant women to establish the reference interval of D-dimer for each trimester [9]. They determined the mean D-dimer levels in the first trimester of 222 ng/mL, in the second of 326 and in the third of 475 ng/ mL. Using 230 ng/mL as the threshold of abnormal, they found that 84% of the reference group in the first trimester had normal D-dimer, 33% in the second, and only 1% in the third trimester. These results are similar to our findings. Kline et al. showed approximately 39% increase in D-dimer concentration for each trimester compared to the previous measurement [8]. A total of 50 women were enrolled in their prospective study. If a cut-off of 0.50 mg/L is used, they found that in the first trimester 50% of women had D-dimer level below cut-off, in the second only 22%, and in the third trimester 0%. Compared to these results, patients who had the D-dimer levels above 0.5 mg/L was lower in our
D-dimer levels during pregnancy
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DOI: 10.3109/14767058.2014.940891
study. However, in their study, 21.8% of subjects had D-dimer levels above 0.5 mg/L in pre-conception. In our study, none of non-pregnant women had D-dimer levels above this conventional value. In another prospective study, findings similar to Kline et al. were declared by Szecsi et al. [10] They found that 25% of pregnant women at 13–20 weeks and all pregnant women at 36–42 weeks have D-dimer levels at or above 0.5 mg/L. The gestational age-related reference intervals of D-dimer calculated by these authors were higher than reported by others. Different D-dimer reference intervals have been observed in different studies. This may be due to different specificity of the antibodies or use of different instruments or different pre-analytical conditions. It may also be important whether the samples analyzed were frozen or not. We analyzed the D-dimer levels in unfrozen samples; however it was analyzed in frozen samples by Szecsi et al. In the present study, plasma fibrinogen levels were also analyzed to determine whether there was a relation between D-dimer and fibrinogen. However, a significant correlation was not identified. In this context, some authors have been declared similar results in their studies [8,10]. In addition, our data showed that 18.6% of pregnant women in first trimester, 29.2% of pregnant women in second trimester, and 42.8% of pregnant women in third trimester had fibrinogen levels that exceeded reference value which was up to 3.8 g/L. Therefore, in order to provide a useful tool in the clinical situations accompanied by altered fibrinogen levels in pregnant women, we calculated the fibrinogen reference intervals as 2.5–4.3, 1.8–5.9 and 2.6–4.9 g/L in the first, second and third trimesters, respectively. The most important limitation of this study is that it is cross-sectional in nature. Therefore, to decrease interindividual variation, further studies that being aimed to determine D-dimer reference interval should be designed as longitudinal study. In conclusion, our finding confirmed the elevation of D-dimer levels from trimester to trimester. In the present study, the established D-dimer reference intervals for each trimester were different from reference interval used in
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common population. These reference intervals may help clinicians to make more accurate clinical decisions. Further clinical studies are needed to determine a clinically useful cut-off point for D-dimer in diagnosis of VTE during pregnancy.
Acknowledgements No funding received. We thank Hu¨seyin Dog˘anay, Evrim ¨ zaydın for their technical assistance. Beyal and Emine O
Declaration of interest The authors report no declarations of interest.
References 1. Breme KA. Haemostatic changes in pregnancy. Best Pract Res Clin Haematol 2003;16:153–68. 2. James AH. Venous thromboembolism in pregnancy. Arterioscler Thromb Vasc Biol 2009;29:326–31. 3. Nijkeuter M, Ginsberg JS, Huisman MV. Diagnosis of deep vein thrombosis and pulmonary embolism in pregnancy: a systematic review. J Thromb Haemost 2006;4:496–500. 4. Chunilal SD, Bates SM. Venous thromboembolism in pregnancy: diagnosis, management and prevention. Thromb Haemost 2009; 101:428–38. 5. Pabinger I, Ay C. Biomarkers and venous thromboembolism. Arterioscler Thromb Vasc Biol 2009;29:332–6. 6. Scarvelis D, Wells PS. Diagnosis and treatment of deep-vein thrombosis. CMAJ 2006;175:1087–92. 7. Morse M. Establishing a normal range for D-dimer levels through pregnancy to aid in the diagnosis of pulmonary embolism and deep vein thrombosis. J Thromb Haemost 2004;2:1202–4. 8. Kline JA, Williams GW, Hernandez-Nino J. D-Dimer concentrations in normal pregnancy: new diagnostic thresholds are needed. Clin Chem 2005;51:825–9. 9. Kovac M, Mikovic Z, Rakicevic L, et al. The use of D-dimer with new cutoff can be useful in diagnosis of venous thromboembolism in pregnancy. Eur J Obstet Gynecol Reprod Biol 2010;148:27–30. 10. Szecsi, PB, Jorgensen M, Klajnbard A, et al. Haemostatic reference intervals in pregnancy. Thromb Haemost 2010;103:718–27. 11. Clinical and Laboratory Standards Institute. Defining, establishing, and verifying reference intervals in the clinical laboratory; approved guideline, 3rd ed. CLSI document C28-A3. Clinical and Laboratory Standards Institute 2008;28(30). 12. Heim SW, Schectman JM, Siadaty MS, Philbrick JT. D-Dimer testing for deep venous thrombosis: a meta-analysis. Clin Chem 2004;50:1136–47.
ORIGINAL ARTICLE
Establishing reference intervals for D-dimer to trimesters S¸ erif Ercan1, Sadullah O¨zkan2, Nihal Yu¨cel3, and Asuman Orc¸un3 1
Department of Clinical Biochemistry, Lu¨leburgaz State Hospital, Kırklareli, Turkey, 2Department of Obstetric and Gynecology, and Department of Clinical Biochemistry, Dr. Lutfi Kirdar Kartal Education and Research Hospital, Istanbul, Turkey
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3
Abstract
Keywords
Objective: When D-dimer is used to evaluate suspected venous thromboembolism in pregnant patients, the reference interval of common population may cause misinterpretation. The present study aims to determine reference intervals of D-dimer in the three trimesters. Methods: Four-hundred sixteen pregnant women and 32 non-pregnant women were enrolled in this cross-sectional study. Reference group had comprised 123 pregnant in the first trimester (5–11 week), 164 pregnant in the second trimester (13–20 week) and 126 pregnant women in the third trimester (25–35 week). D-dimer levels were analyzed via immunoturbidimetric assay. Results: If the threshold of 0.50 mg/L for diagnosis of VTE is used, 4.8% of pregnant women in the second trimester and 23.8% of pregnant women in the third trimester would have D-dimer levels exceeding this cut-off value. Reference intervals of D-dimer were determined as 0.11–0.40 mg/L; 0.14–0.75 mg/L and 0.16–1.3 mg/L in first, second and third trimester, respectively. Conclusion: The established D-dimer reference intervals for each trimester of pregnancy are different from those used in common population. These reference intervals may assist clinicians in making accurate clinical decisions. Further studies are needed to establish new cut-off values for the D-dimer to rule out VTE in each trimester.
D-dimer, pregnancy, reference interval, trimesters, venous thromboembolism
Introduction Normal pregnancy is a hypercoagulable state because of increased concentration of procoagulants, decreased levels of anticoagulants and diminished fibrinolytic activity [1]. The hypercoagulable state protects pregnant women from the bleeding complications of miscarriage and childbirth. However, the risk of venous thromboembolism (VTE) is increased 4- to 5-fold in pregnant women compared to women who are not pregnant. The overall prevalence of thromboembolic events during pregnancy is approximately 2 per 1000 deliveries. VTE accounts for 1.1 deaths per 100 000 deliveries, or 10% of all maternal deaths [2]. Diagnosis of VTE in pregnancy is challenging due to many of the classic symptoms such as leg swelling and respiratory symptoms which are also associated with normal pregnancy [3]. In diagnostic imaging of deep venous thrombosis (DVT), contrast venography or compression ultrasonography has important limitations. Venography is invasive, expensive and exposes the patient to the radiation. Although compression ultrasound has high diagnostic sensitivity and specificity for
Address for correspondence: S ¸ erif Ercan, Department of Clinical ¨ zeri Yeni Biochemistry, Lu¨leburgaz State Hospital, D 100 Karayolu U Sanayi Kar¸s ısı 39750 Lu¨leburgaz/Kırklareli, Istanbul, Turkey. Tel: +90 5555875287. Fax: +90 2884131046. E-mail: serifercan@ yahoo.com.tr
History Received 4 July 2013 Revised 15 June 2014 Accepted 30 June 2014 Published online 25 July 2014
DVT without ionizing radiation, it is not always available in many clinics and small hospitals. In addition, it has poor sensitivity for detection of calf vein thrombosis, and sequential testing is often needed when the initial scan is negative. In the investigation of suspected pulmonary emboli (PE), all available imaging techniques such as scintillation ventilation-perfusion lung scan or a computed tomography angiography cause exposure of both the mother and fetus to the effects of ionizing radiation [3,4]. To diagnose the VTE in pregnancy; objective, rapid, inexpensive, non-invasive and harmless fetus tests are required. D-dimer, a degradation product of a cross-linked fibrin blood clot, has the potential to detect thrombosis in any part of the venous system. A negative value of D-dimer may safely rule out both DVT and PE with a high sensitivity up to 95% and a negative predictive value of almost 100% [5]. D-dimer is sensitive but non-specific marker for VTE because it is elevated in variety of non-thrombotic conditions such as recent major surgery, hemorrhage, trauma, cancer and pregnancy [6]. D-dimer levels increase progressively from trimester to trimester during normal pregnancy [7–10]. When the D-dimer cut-off value for common population is used to evaluate VTE in pregnant women, this increase may cause misinterpretation. The D-dimer assay has been suggested as an initial test to rule out VTE and reduce the number of patients requiring diagnostic imaging. However, D-Dimer role in algorithms for
2
S¸ . Ercan et al.
diagnosis of VTE in pregnancy has not been adequately studied. Moreover, D-dimer reference values derived from sufficient number of healthy pregnant women for different trimesters are not available. Therefore, prior to clinical application, we aimed in this study, to establish D-Dimer reference values for each trimester.
J Matern Fetal Neonatal Med Downloaded from informahealthcare.com by Columbia University on 03/01/15 For personal use only.
Methods This cross-sectional study included 448 healthy women including 416 pregnant and 32 non-pregnant women. Pregnant women followed by Dr. Lutfi Kirdar Kartal Training and Research Hospital, Department of Obstetrics and Gynecology were enrolled in the study. According to recommendation of International Federation of Clinical Chemistry and Clinical and Laboratory Standards Institute [11], healthy pregnant women for reference population were selected. The exclusion criteria were designed to minimize any possible influence on D-dimer levels: any personal history of thromboembolic disease, bleeding disorders, liver disease (abnormal liver function tests), renal disease (abnormal creatinine), autoimmune diseases such as systemic lupus erythematosus, pancreatitis (abnormal amylase concentrations with abdominal pain), morbid obesity (body mass index 40 kg/m2), a condition requiring daily injections (such as insulin-dependent diabetes mellitus) and current infection with fever 438 C. The additional exclusion criteria were defined as diastolic blood pressure 485 mmHg and proteinuria. This study has been carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans. The pregnant women were divided into three groups based on gestational age: first trimester (5–11 weeks, n ¼ 123), second trimester (13–20 weeks, n ¼ 166), and third trimester (25–35 weeks, n ¼ 127). Gestational age was determined by ultrasonographic biometric examination and the date of last menstrual period. When discrepancy between ultrasound and last menstrual period was higher than a week, gestational age was determined according to biometric values. A control group comprised age-matched healthy nonpregnant women were selected according to exclusion criteria used in pregnant group. Blood samples were obtained in follicular phase of menstruation cycle. Blood samples were drawn into a tube containing 0.11 mmol/L (3.2%) sodium citrate (one part anticoagulant plus nine parts blood). Plasma was prepared by centrifugation at 2000g for 10-min. D-dimer levels were measured in fresh, unfrozen plasma samples. Prothrombin time (PT), activated partial thromboplastin time (aPTT) and fibrinogen levels were also analyzed in these plasma samples. Analyses were performed within 4 hours of blood sampling. D-Dimer analysis was performed using the AMAX AUTO D-Dimer kit (Trinity Biotech Plc, Co Wicklow, Ireland) on the AMAX 400 automated coagulation analyzer (Trinity Biotech Plc). This analysis is based on immunoturbidimetric method. By conventional mechanic coagulometric techniques on the same coagulometer, PT, aPTT and fibrinogen levels were analyzed using TriniCLOT PT HTF, TriniCLOT aPTT HS and MDA Fibriquik reagents (Trinity Biotech Plc, Co Wicklow, Ireland), respectively.
J Matern Fetal Neonatal Med, Early Online: 1–5
Statistical analyses were performed using Statistical Package for Social Sciences (SPSS 15.0, SPSS Inc., Chicago, IL) in computerized media. Variables are presented as means ± SD and median according to data distribution. The significance of the mean differences was analyzed with Kruskal–Wallis test. If the Kruskal–Wallis test revealed significant differences, pair-wise comparisons with MannWhitney Test were used to identify the differences. p Values less than or equal to 0.05 were considered to be statistically significant. The MedCalc program (version 12.2.1.0) was used to calculate the non-parametric 2.5–97.5 percentile reference intervals for D-dimer. Reed method was used to determine outliers of data sets.
Results In this study, 416 pregnant women and 32 non-pregnant women were included. Two pregnant women were excluded from study because of abnormal PT results in the first and the second trimester groups. One pregnant woman was also excluded because of elevated aPTT result in the second trimester group. Reference population had finally comprised 123 pregnant in the first trimester (5–11 week), 164 pregnant in the second trimester (13–20 week) and 126 pregnant women in the third trimester (25–35 week). Median age of pregnant women was 27 years (ranging from 16 to 45 years), and that of non-pregnant women was 25 years (ranging from 17 to 35 years). There was no significant difference. The mean ± SD or median values of each parameter measured in pregnant and non-pregnant women are shown in Table 1. As compared to non-pregnant women, D-dimer and fibrinogen levels were found to be significantly elevated in each trimester (Figures 1 and 2, respectively; Table 1). In addition, aPTT was significantly shortened (p50.01) and PT was not changed (Table 1). D-dimer levels had significantly higher in the second trimester than those of the first trimester (median: 0.29 versus 0.18 mg/L, p50.001). D-dimer levels in the third trimester were also found to be significantly increased compared to the second trimester (median: 0.31 versus 0.29 mg/L, p50.001; Table 1). There was no significant difference of fibrinogen levels between the first and the second trimesters. However, fibrinogen levels in the third trimester were higher than those of the second trimester (median: 3.7 versus 3.4 g/L, p50.01; Table 1). There was no significant difference of PT values between the first and the second trimesters and between the second and the third trimesters. aPTT values in the second trimester were found to be elevated compared to the first trimester (p50.001), however there was no significant difference of aPTT values between the second and the third trimesters (Table 1). Reference intervals for PT and aPTT are 11.1–13.2 s and 22.6–35 s, respectively. PT and aPTT values were found to be within the reference intervals in almost all of the
D-dimer levels during pregnancy
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DOI: 10.3109/14767058.2014.940891
pregnant women. Two of the pregnant women had slightly elevated PT INR values. Only one pregnant woman had slightly elevated aPTT values. Twenty-three pregnant women in the first trimester, 48 pregnant women in the second trimester and 54 pregnant women in the third trimester had values that exceeded the upper limit of fibrinogen which was 3.8 g/L (Figure 2). In our laboratory, the upper limit for D-dimer used in common population was 0.15 mg/L. The control group fell below our current upper limit. Moreover, in accordance with the upper limit of common population, the upper limit for D-dimer for non-pregnant women was determined as 0.16 mg/L. D-dimer values of 117 of the pregnants in the first trimester, 162 of the pregnants in the second trimester and all the pregnants in the third trimester exceeded the upper limit of D-dimer. When the cut-off of 0.50 mg/L was used in diagnosis Table 1. The D-dimer, fibrinogen, PT and aPTT values in healthy pregnant and non-pregnant women.
Control n 32 D-dimer (mg/L) Median 0.12 Range 0.06–0.14 Fibrinogen (g/L) Median 3.1 Range 1.8–3.8 PT (s) Mean ±SD 11.9 ± 1.4 aPTT (s) Mean ± SD 34.2 ± 2.7
1st Trimester
2nd Trimester
3rd Trimester
123
164
126
0.18* 0.10–0.46
0.29*,** 0.10–1.46
0.31*,**,*** 0.13–1.75
3.5* 2.1–4.3
3.4* 1.5–7.1
3.7*,*** 2.2–5.9
12 ± 1.6
11.9 ± 1.6
11.8 ± 1.6
32.5 ± 3.4ô
30.5 ± 2.7ô,**
30.5 ± 2.8ô
*p50.001 versus control; **p50.001 versus 1st trimester; ***p50.001 versus 2nd trimester; ôp50.01 versus control.
Figure 1. Plasma D-dimer levels in healthy pregnant and non-pregnant women. Vertical point plot shows the D-dimer levels for each subject. The smooth horizontal bar indicates the cut-off value for VTE. Interrupted horizontal bar presents the upper limit of reference interval for non-pregnant women.
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of VTE, 8 the pregnant women in the second trimester and 30 pregnant women in the third trimester were found to exceed this threshold (Figure 1). By correlation analysis, we aimed to determine whether the increase in fibrinogen was correlated with the increase in D-dimer. A significant correlation was not identified between D-dimer and fibrinogen levels. The calculated reference intervals for D-dimer are presented in Table 2. The reference interval of D-dimer was established as 0.11–0.40, 0.14–0.75 and 0.16–1.3 mg/L in the first, second and third trimesters, respectively. For fibrinogen, we also calculated the reference interval as 2.5–4.3, 1.8–5.9 and 2.6–4.9 g/L in the first, second and third trimesters, respectively.
Discussion D-Dimer testing is quick, simple, and inexpensive and it has a high sensitivity of up to 95% and a negative predictive value of nearly 100% for VTE [5,12]. Therefore, D-dimer testing should be used to rule out VTE in order to reduce the number of patients requiring potentially harmful diagnostic imaging. Pregnant women have a two-to-five fold higher risk of VTE compared to non-pregnant women. The diagnosis of VTE during pregnancy is difficult due to physiologic changes and the possibility of differences in pathophysiology and presentation of VTE in pregnancy [3]. Although D-dimer testing has an important role in the exclusion of VTE in the non-pregnant population, D-Dimer incorporation in algorithms for diagnosing VTE in pregnant women has not been adequately studied. Furthermore, there is not adequate number of studies aimed to establish the reference values of D-dimer with a sufficient number of pregnant women for each trimester. Previous studies demonstrated that plasma D-dimer levels rise during pregnancy [7–10]. As expected, we found the
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S¸ . Ercan et al.
J Matern Fetal Neonatal Med, Early Online: 1–5
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Figure 2. Plasma fibrinogen levels in healthy pregnant and non-pregnant women. Vertical point plot shows the fibrinogen levels for each subject. The horizontal bar indicates the upper limit of reference interval for non-pregnant women.
Table 2. The reference intervals of D-dimer and fibrinogen to trimesters and non-pregnant women.
D-Dimer (mg/L) Reference interval method 95% Reference intervals Fibrinogen (g/L) Reference interval method 95% Reference intervals
Controls
First trimester
Second trimester
Third trimester
Parametric 0.07–0.16
Non-parametric 0.11–0.40
Non-parametric 0.14–0.75
Non-parametric 0.16–1.3
Parametric 2.2–3.9
Parametric 2.5–4.3
Non-parametric 1.8–5.9
Parametric 2.6–4.9
increase of the D-dimer concentration during pregnancy from trimester to trimester. The highest D-dimer levels were obtained during the third trimester. We also determined that 95.1% of pregnant women in the first trimester, 98.7% of pregnant women in the second trimester, 100% of pregnant women in the third trimester had D-dimer levels that exceeded reference value which was up to 0.15 mg/L. If the threshold of 0.50 mg/L for diagnosis of VTE is used, 4.8% of pregnant women in the second trimester and 23.8% of pregnant women in the third trimester were found to exceed this cut-off value. The elevation of D-dimer levels from trimester to trimester restricts its use in the evaluation of suspected VTE in pregnant women. Determination of a cut-off value for VTE during pregnancy is difficult because it requires very expensive multicenter study which includes large numbers of pregnant women tested for VTE. Therefore, prior to clinical application, the determination of reference intervals of D-dimer for each trimester in a sufficient number of reference individuals is essential. International Federation of Clinical Chemistry and Clinical and Laboratory Standards Institute guidelines recommend establishing a reference interval in at least 120 reference individuals using the non-parametric ranking method [11]. Therefore, at least 120 pregnant women for each trimester were included into our study. By non-parametric method, we
determined the reference interval of D-dimer as: for the first trimester 0.11–0.40 mg/L; for the second trimester 0.14–0.75 mg/L and for the third trimester 0.16–1.3 mg/L. In a prospective study, Morse analyzed D-dimer levels throughout the pregnancy in 48 pregnant women [7]. Author calculated the reference interval of D-dimer as mean ± SD, which were 191 ± 25 ng/mL in 16 weeks, 393 ± 72 ng/mL in 26 weeks, 544 ± 96 ng/mL in 34 weeks and 140 ± 58 ng/mL in controls. Recently, Kovac et al. prospectively analyzed D-dimer levels in 89 healthy pregnant women to establish the reference interval of D-dimer for each trimester [9]. They determined the mean D-dimer levels in the first trimester of 222 ng/mL, in the second of 326 and in the third of 475 ng/ mL. Using 230 ng/mL as the threshold of abnormal, they found that 84% of the reference group in the first trimester had normal D-dimer, 33% in the second, and only 1% in the third trimester. These results are similar to our findings. Kline et al. showed approximately 39% increase in D-dimer concentration for each trimester compared to the previous measurement [8]. A total of 50 women were enrolled in their prospective study. If a cut-off of 0.50 mg/L is used, they found that in the first trimester 50% of women had D-dimer level below cut-off, in the second only 22%, and in the third trimester 0%. Compared to these results, patients who had the D-dimer levels above 0.5 mg/L was lower in our
D-dimer levels during pregnancy
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DOI: 10.3109/14767058.2014.940891
study. However, in their study, 21.8% of subjects had D-dimer levels above 0.5 mg/L in pre-conception. In our study, none of non-pregnant women had D-dimer levels above this conventional value. In another prospective study, findings similar to Kline et al. were declared by Szecsi et al. [10] They found that 25% of pregnant women at 13–20 weeks and all pregnant women at 36–42 weeks have D-dimer levels at or above 0.5 mg/L. The gestational age-related reference intervals of D-dimer calculated by these authors were higher than reported by others. Different D-dimer reference intervals have been observed in different studies. This may be due to different specificity of the antibodies or use of different instruments or different pre-analytical conditions. It may also be important whether the samples analyzed were frozen or not. We analyzed the D-dimer levels in unfrozen samples; however it was analyzed in frozen samples by Szecsi et al. In the present study, plasma fibrinogen levels were also analyzed to determine whether there was a relation between D-dimer and fibrinogen. However, a significant correlation was not identified. In this context, some authors have been declared similar results in their studies [8,10]. In addition, our data showed that 18.6% of pregnant women in first trimester, 29.2% of pregnant women in second trimester, and 42.8% of pregnant women in third trimester had fibrinogen levels that exceeded reference value which was up to 3.8 g/L. Therefore, in order to provide a useful tool in the clinical situations accompanied by altered fibrinogen levels in pregnant women, we calculated the fibrinogen reference intervals as 2.5–4.3, 1.8–5.9 and 2.6–4.9 g/L in the first, second and third trimesters, respectively. The most important limitation of this study is that it is cross-sectional in nature. Therefore, to decrease interindividual variation, further studies that being aimed to determine D-dimer reference interval should be designed as longitudinal study. In conclusion, our finding confirmed the elevation of D-dimer levels from trimester to trimester. In the present study, the established D-dimer reference intervals for each trimester were different from reference interval used in
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common population. These reference intervals may help clinicians to make more accurate clinical decisions. Further clinical studies are needed to determine a clinically useful cut-off point for D-dimer in diagnosis of VTE during pregnancy.
Acknowledgements No funding received. We thank Hu¨seyin Dog˘anay, Evrim ¨ zaydın for their technical assistance. Beyal and Emine O
Declaration of interest The authors report no declarations of interest.
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