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Reference interval of D-dimer in pregnant women☆
Clinical Biochemistry 34 (2001) 331–333
Reference interval of D-dimer in pregnant women夞 Davide Giavarinaa,*, Gabriella Mezzenaa, Romolo M. Dorizzib, Giuliano Soffiatia a
Laboratorio di Chimica clinica ed Ematologia, Ospedale S. Bortolo, via Rodolfi 37, 36100, Vicenza, Italy b Laboratorio Analisi Chimico-Cliniche ed Ematologiche, Azienda Ospedaliera di Verona, Italy Received 9 January 2001; received in revised form 19 March 2001; accepted 20 March 2001
1. Introduction Thromboembolism is the leading cause of maternal mortality accounting for almost 20% of maternal mortality in UK. The physiological or pathophysiological activation of hemostasis during pregnancy results in the generation of the so-called activation markers, reflecting hypercoagulability and therefore representing an imbalance in the hemostatic system [1]. During normal pregnancy, and particularly in the third trimester, the concentration of several blood coagulation factors (VII, VIII, IX and X) and of fibrinogen (its 50% rise contributes to the increase in erythrocytes sedimentation rate) increases. Many authors reported high concentration of the so-called D-dimer, a product of plasminmediated proteolysis of fibrin. Thrombus formation begins when fibrinogen is converted to fibrin by thrombin and highly crosslinked fibrin monomers are the major structural component of the resultant thrombus. The crosslinking of fibrin generates unique antigenic determinants, one of which is the bond between two D domains of adjacent fibrin monomers [2]. D-dimer concentration increases in many pathologies in which fibrin is degraded by plasmin, including deep venous thrombosis and acute venous thromboembolism; therefore its specificity for deep venous thrombosis is low while sensitivity and negative predictive value are high. D-dimer is commonly measured by methods based on monoclonal antibodies: enzyme-linked immunoassays (ELISA), latex agglutination and whole blood agglutination [2]. Accuracy studies demonstrated the high sensitivity and the low practicability on stat basis of conventional ELISAs. Brill-Edwards and Lee recently concluded that results from one manufacturer’s assay are often not applicable to assays
夞 Part of the data have been presented as poster at the 52nd National Meeting of American Association of Clinical Chemistry, S. Francisco, USA, July 23–27, 2000. * Corresponding author. E-mail address: [email protected] (G.M.R.).
of other manufacturers and different laboratories may obtain different results even when use the same manufacturer’s method [2]. Notwithstanding the relevance and the complexity of the production of proper D-Dimer reference intervals in pregnant women, only a few studies have been devoted to the problem. In 1991 van Wersch et al, using an ELISA method, reported a significant increase of the D-dimer concentration with gestational age, with almost 80% of the values above the upper limit of the reference interval (450 g/L) [3]. Francalanci et al and Ghirardini et al confirmed this finding reporting a progressive increase in D-dimer concentration during normal pregnancy [4 – 6]. Nolan et al reported that D-dimer test, measured using a rapid latex agglutination slide test, conferred sensitivity, specificity, positive predictive value, and negative predictive value of 67%, 93%, 91%, and 48%, respectively, and concluded that the D-dimer slide test may be a superior method for early diagnosing abruptio placentae [7]. An excessive hypercoagulable state, as suggested by a significant increase of D-dimer concentration, in patients with severe preeclampsia may be associated with the termination of pregnancy [8]. Finally, Padden recently stated that the D-dimer test might be useful for early identification of patients with preeclampsia who are developing severe HELLP syndrome [9]. The aim of this study was to calculate, following the International Federation of Clinical Chemistry recommendations, the reference interval of D-Dimer throughout uncomplicated pregnancies. 2. Materials and methods We measured plasma concentration of D-Dimer in 340 consecutive uncomplicated pregnant women aged 18 to 41 years. All the women were healthy; in particular none had hypertension, diabetes mellitus or other pathology. None had a history of previous abnormal pregnancy, abortion or hemostatic disease. The assays were carried out on fresh plasma within 2 hours after sample collection using a latex
0009-9120/01/$ – see front matter © 2001 The Canadian Society of Clinical Chemists. All rights reserved. PII: S 0 0 0 9 - 9 1 2 0 ( 0 1 ) 0 0 2 0 5 - 3
332
D. Giavarina et al. / Clinical Biochemistry 34 (2001) 331–333
years, between 21st and 40th gestational week (group B). The subjects were informed about the purpose of the investigation and gave their informed consent. 2.1. Statistical analysis
Fig. 1. Relationship between D-dimer plasma levels and gestational age.
agglutination method and an automated analyzer (STALiatest™, Diagnostica Stago, Roche, Basel, Switzerland). One hundred ninety eight women, aged (mean ⫾ SD) 30.6 ⫾ 4.1 years, were studied between 1st and 20th gestational week (group A), and 142 women, aged 30.3 ⫾ 5.7
Spearman’s correlation coefficient between D-dimer concentration and gestational week and Student’s test analysis of the difference were calculated using the software StatsDirect (CamCode, Ashwell, UK). In both groups the hypothesis that the data fit a Gaussian distribution was rejected at the 0.05 significance using the KolmogorovSmirnov test (Dmax: group A ⫽ 0.117; group B ⫽ 0.069). Kolmogorov-Smirnov test and reference values were calculated using Refval, a software kindly provided by HE Solberg [10]. No extreme values were identified as an outlier according to the Dixon test [10].
3. Results A highly significant positive correlation was observed between gestational age and D-Dimer concentration (r⫽
Fig. 2. D-dimer plasma levels in gestational weeks 1st–20th and 21st– 40th.
D. Giavarina et al. / Clinical Biochemistry 34 (2001) 331–333
0.509; p ⬍ 0.0001) (Fig. 1). The D-dimer concentration of the subjects in the two groups is shown in Fig. 2. In group B, D-dimer levels were higher than in group A (p ⬍ 0.0001). The group B was further divided in 2 groups: one comprising women in 21st-30th gestational weeks (group 2A) and one comprising women in 31st-40th gestational weeks (group 2B). The mean concentration was respectively 880 /L and 1090 /L, the 97.5% percentile 3100 /L and 3210 /L and the maximum concentration was respectively 3600 /L and 4300 /L. The nonparametric estimation of the reference interval (2.5–97.5 percentiles) is: group A: ⬍220 to 1275 /L (0.90 confidence interval: 1060 –2240); group B: ⬍220 to 3130 /L (0.90 confidence interval: 2070 – 4300). 4. Discussion The measurement of D-dimer has been proposed in pregnant women for excluding of a deep venous thrombosis and for the diagnosis of severe pregnancy complications. The interpretation of an observed value in the proper diagnostic context is subordinate to the definition of appropriate reference intervals and decision limits. The misunderstanding of the concept of D-dimer reference values and decision limits led laboratory professionals and clinicians to misinterpret the results strongly reducing their potential information. The adoption of reference intervals obtained in notpregnant women greatly reduces the specificity of the assay and leads to the misclassification of 80% of all the pregnant women who present high D-dimer levels since pregnancy is an hypercoagulable state by itself and an important risk factor for thrombosis. Moreover different methods for Ddimer assay can yield much different results and therefore transferring the reference interval among different methods, laboratories and geographical areas represents a wrong and dangerous practice [2]. The present study demonstrates that D-Dimer concentration rises during pregnancy and there-
333
fore the proper reference interval must be extracted in an adequate number of healthy pregnant women of different gestational weeks. To our knowledge until now only a restricted number of subjects have been studied. Van Wersch et al. studied 56 pregnant women (gestational age: 6 – 40 weeks) [3] and Francalanci et al. 63 (21 before 20th weeks, 20 between 21st and 30th and 22 between 31st and 40th) [6]. A proper estimation or verification of D-Dimer reference interval in pregnancy is very advisable for laboratorians and clinicians.
References [1] Dati F, Pelzer H, Wagner C. Relevance of markers of hemostasis activation in obstetrics/gynecology and pediatrics. Semin Thromb Hemost 1998;24:443– 8. [2] Brill-Edwards, Lee A. D-dimer testing in the diagnosis of acute venous thromboembolism. Thromb Haemost 1999;82:688 –94. [3] van Wersch JWJ, Ubachs JMH. Blood coagulation and fibrinolysis during normal pregnancy. Eur J Clin Chem Clin Biochem 1991;29: 45–50. [4] Francalanci I, Comeglio P, Liotta AA et al. D-Dimer in intra-uterine growth retardation, and gestational hypertension. Thromb Res 1995; 80:89 –92. [5] Ghirardini G, Bottioni M, Bertellini C. D-dimer after delivery in uncomplicated pregnancies. Clin Exp Obstet Gynecol 1999;26: 211–2. [6] Francalanci I, Comeglio P, Alessandrello Liotta A, et al. D-dimer plasma levels during normal pregnancy measured by specific ELISA. Int J Clin Lab Res 1997;27:65–7. [7] Nolan TE, Smith RP, Devoe LD. A rapid test for abruptio placentae: evaluation of a D-dimer latex agglutination slide test. Am J Obstet Gynecol 1993;169:265– 8. [8] Kobayashi T, Tokunaga T, Sugimura M, et al. Coagulation/fibrinolysis disorder in patients with severe preeclampsia. Semin Thromb Hemost 1999;25:451– 4. [9] Padden MO. HELLP syndrome: recognition, and perinatal management. Am Fam Physician 1999;60:829 –39. [10] Solberg HE. Establishment and use of reference values. In: Burtis CA, Ashwood ER, editors. Tietz textbook of clinical chemistry. 2nd ed. Saunders: Philadelphia, 1994. p.454 – 84.
Reference interval of D-dimer in pregnant women夞 Davide Giavarinaa,*, Gabriella Mezzenaa, Romolo M. Dorizzib, Giuliano Soffiatia a
Laboratorio di Chimica clinica ed Ematologia, Ospedale S. Bortolo, via Rodolfi 37, 36100, Vicenza, Italy b Laboratorio Analisi Chimico-Cliniche ed Ematologiche, Azienda Ospedaliera di Verona, Italy Received 9 January 2001; received in revised form 19 March 2001; accepted 20 March 2001
1. Introduction Thromboembolism is the leading cause of maternal mortality accounting for almost 20% of maternal mortality in UK. The physiological or pathophysiological activation of hemostasis during pregnancy results in the generation of the so-called activation markers, reflecting hypercoagulability and therefore representing an imbalance in the hemostatic system [1]. During normal pregnancy, and particularly in the third trimester, the concentration of several blood coagulation factors (VII, VIII, IX and X) and of fibrinogen (its 50% rise contributes to the increase in erythrocytes sedimentation rate) increases. Many authors reported high concentration of the so-called D-dimer, a product of plasminmediated proteolysis of fibrin. Thrombus formation begins when fibrinogen is converted to fibrin by thrombin and highly crosslinked fibrin monomers are the major structural component of the resultant thrombus. The crosslinking of fibrin generates unique antigenic determinants, one of which is the bond between two D domains of adjacent fibrin monomers [2]. D-dimer concentration increases in many pathologies in which fibrin is degraded by plasmin, including deep venous thrombosis and acute venous thromboembolism; therefore its specificity for deep venous thrombosis is low while sensitivity and negative predictive value are high. D-dimer is commonly measured by methods based on monoclonal antibodies: enzyme-linked immunoassays (ELISA), latex agglutination and whole blood agglutination [2]. Accuracy studies demonstrated the high sensitivity and the low practicability on stat basis of conventional ELISAs. Brill-Edwards and Lee recently concluded that results from one manufacturer’s assay are often not applicable to assays
夞 Part of the data have been presented as poster at the 52nd National Meeting of American Association of Clinical Chemistry, S. Francisco, USA, July 23–27, 2000. * Corresponding author. E-mail address: [email protected] (G.M.R.).
of other manufacturers and different laboratories may obtain different results even when use the same manufacturer’s method [2]. Notwithstanding the relevance and the complexity of the production of proper D-Dimer reference intervals in pregnant women, only a few studies have been devoted to the problem. In 1991 van Wersch et al, using an ELISA method, reported a significant increase of the D-dimer concentration with gestational age, with almost 80% of the values above the upper limit of the reference interval (450 g/L) [3]. Francalanci et al and Ghirardini et al confirmed this finding reporting a progressive increase in D-dimer concentration during normal pregnancy [4 – 6]. Nolan et al reported that D-dimer test, measured using a rapid latex agglutination slide test, conferred sensitivity, specificity, positive predictive value, and negative predictive value of 67%, 93%, 91%, and 48%, respectively, and concluded that the D-dimer slide test may be a superior method for early diagnosing abruptio placentae [7]. An excessive hypercoagulable state, as suggested by a significant increase of D-dimer concentration, in patients with severe preeclampsia may be associated with the termination of pregnancy [8]. Finally, Padden recently stated that the D-dimer test might be useful for early identification of patients with preeclampsia who are developing severe HELLP syndrome [9]. The aim of this study was to calculate, following the International Federation of Clinical Chemistry recommendations, the reference interval of D-Dimer throughout uncomplicated pregnancies. 2. Materials and methods We measured plasma concentration of D-Dimer in 340 consecutive uncomplicated pregnant women aged 18 to 41 years. All the women were healthy; in particular none had hypertension, diabetes mellitus or other pathology. None had a history of previous abnormal pregnancy, abortion or hemostatic disease. The assays were carried out on fresh plasma within 2 hours after sample collection using a latex
0009-9120/01/$ – see front matter © 2001 The Canadian Society of Clinical Chemists. All rights reserved. PII: S 0 0 0 9 - 9 1 2 0 ( 0 1 ) 0 0 2 0 5 - 3
332
D. Giavarina et al. / Clinical Biochemistry 34 (2001) 331–333
years, between 21st and 40th gestational week (group B). The subjects were informed about the purpose of the investigation and gave their informed consent. 2.1. Statistical analysis
Fig. 1. Relationship between D-dimer plasma levels and gestational age.
agglutination method and an automated analyzer (STALiatest™, Diagnostica Stago, Roche, Basel, Switzerland). One hundred ninety eight women, aged (mean ⫾ SD) 30.6 ⫾ 4.1 years, were studied between 1st and 20th gestational week (group A), and 142 women, aged 30.3 ⫾ 5.7
Spearman’s correlation coefficient between D-dimer concentration and gestational week and Student’s test analysis of the difference were calculated using the software StatsDirect (CamCode, Ashwell, UK). In both groups the hypothesis that the data fit a Gaussian distribution was rejected at the 0.05 significance using the KolmogorovSmirnov test (Dmax: group A ⫽ 0.117; group B ⫽ 0.069). Kolmogorov-Smirnov test and reference values were calculated using Refval, a software kindly provided by HE Solberg [10]. No extreme values were identified as an outlier according to the Dixon test [10].
3. Results A highly significant positive correlation was observed between gestational age and D-Dimer concentration (r⫽
Fig. 2. D-dimer plasma levels in gestational weeks 1st–20th and 21st– 40th.
D. Giavarina et al. / Clinical Biochemistry 34 (2001) 331–333
0.509; p ⬍ 0.0001) (Fig. 1). The D-dimer concentration of the subjects in the two groups is shown in Fig. 2. In group B, D-dimer levels were higher than in group A (p ⬍ 0.0001). The group B was further divided in 2 groups: one comprising women in 21st-30th gestational weeks (group 2A) and one comprising women in 31st-40th gestational weeks (group 2B). The mean concentration was respectively 880 /L and 1090 /L, the 97.5% percentile 3100 /L and 3210 /L and the maximum concentration was respectively 3600 /L and 4300 /L. The nonparametric estimation of the reference interval (2.5–97.5 percentiles) is: group A: ⬍220 to 1275 /L (0.90 confidence interval: 1060 –2240); group B: ⬍220 to 3130 /L (0.90 confidence interval: 2070 – 4300). 4. Discussion The measurement of D-dimer has been proposed in pregnant women for excluding of a deep venous thrombosis and for the diagnosis of severe pregnancy complications. The interpretation of an observed value in the proper diagnostic context is subordinate to the definition of appropriate reference intervals and decision limits. The misunderstanding of the concept of D-dimer reference values and decision limits led laboratory professionals and clinicians to misinterpret the results strongly reducing their potential information. The adoption of reference intervals obtained in notpregnant women greatly reduces the specificity of the assay and leads to the misclassification of 80% of all the pregnant women who present high D-dimer levels since pregnancy is an hypercoagulable state by itself and an important risk factor for thrombosis. Moreover different methods for Ddimer assay can yield much different results and therefore transferring the reference interval among different methods, laboratories and geographical areas represents a wrong and dangerous practice [2]. The present study demonstrates that D-Dimer concentration rises during pregnancy and there-
333
fore the proper reference interval must be extracted in an adequate number of healthy pregnant women of different gestational weeks. To our knowledge until now only a restricted number of subjects have been studied. Van Wersch et al. studied 56 pregnant women (gestational age: 6 – 40 weeks) [3] and Francalanci et al. 63 (21 before 20th weeks, 20 between 21st and 30th and 22 between 31st and 40th) [6]. A proper estimation or verification of D-Dimer reference interval in pregnancy is very advisable for laboratorians and clinicians.
References [1] Dati F, Pelzer H, Wagner C. Relevance of markers of hemostasis activation in obstetrics/gynecology and pediatrics. Semin Thromb Hemost 1998;24:443– 8. [2] Brill-Edwards, Lee A. D-dimer testing in the diagnosis of acute venous thromboembolism. Thromb Haemost 1999;82:688 –94. [3] van Wersch JWJ, Ubachs JMH. Blood coagulation and fibrinolysis during normal pregnancy. Eur J Clin Chem Clin Biochem 1991;29: 45–50. [4] Francalanci I, Comeglio P, Liotta AA et al. D-Dimer in intra-uterine growth retardation, and gestational hypertension. Thromb Res 1995; 80:89 –92. [5] Ghirardini G, Bottioni M, Bertellini C. D-dimer after delivery in uncomplicated pregnancies. Clin Exp Obstet Gynecol 1999;26: 211–2. [6] Francalanci I, Comeglio P, Alessandrello Liotta A, et al. D-dimer plasma levels during normal pregnancy measured by specific ELISA. Int J Clin Lab Res 1997;27:65–7. [7] Nolan TE, Smith RP, Devoe LD. A rapid test for abruptio placentae: evaluation of a D-dimer latex agglutination slide test. Am J Obstet Gynecol 1993;169:265– 8. [8] Kobayashi T, Tokunaga T, Sugimura M, et al. Coagulation/fibrinolysis disorder in patients with severe preeclampsia. Semin Thromb Hemost 1999;25:451– 4. [9] Padden MO. HELLP syndrome: recognition, and perinatal management. Am Fam Physician 1999;60:829 –39. [10] Solberg HE. Establishment and use of reference values. In: Burtis CA, Ashwood ER, editors. Tietz textbook of clinical chemistry. 2nd ed. Saunders: Philadelphia, 1994. p.454 – 84.