University Of Khartoum
Faculty of Medical Laboratory Sciences
Haematology Department
Risk factors for thrombophilia in expatriate pregnant Sudanese women in
Eastern Saudi Arabia.
Siddig Safiddin Mohammed.
(B.Sc, U. of Khartoum)
A thesis submitted for the fulfillment of Master degree in Medical Laboratory
Sciences (Haematology)
LIST OF CONTENTS
Subject
List of contents
Dedication
Acknowledgements
Abstract (Arabic)
Abstract (English)
List of Abbreviation
Objectives, Rationale & hypothesis
Page N0.
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Chapter I
1.0 Introduction
1.1 Definition
1.1.1 Vascular endothelium
1.1.2 Platelet system
1.1.3 Coagulation system
1.1.4 Natural inhibitors
1.1.5 Fibrinolytic system
1.2 Classification of thrombophilias
1.2.1 Congenital thrombophilia
1.2.2 Acquired thrombophilia
1.3 Epidemiology of maternal venous thromboembolism
1.4 Etiology of maternal thrombosis & causes of natural
anticoagulants deficiencies
1.4.1 Antithrombin III (ATIII) deficiency
1.4.2 Protein C (PC) deficiency
1.4.3 Protein S (PS) deficiency
1.4.4 Factor V Leiden (FVL)
1.4.5 Prothrombin 20210
1.4.6 Mehtylenetetrahydrofolate reductase deficiency (MTHFR)
1.4.7 Myloproliferative disorders (MPD)
1.4.8 Antiphospholipid antibodies
1.4.9 Diagnosis of venous thrombosis
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Chapter II
2.0 Materials and methods
2.1 Study design
2.2 Patients and Samples
2.2.1 Patients inclusion criteria
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Chapter II Cont.
2.2.2 Controls inclusion criteria
2.2.3 Samples
2.2.4 Serum Preperation
2.2.5 Preperation of citrated plasma for coagulation studies
2.2.6 EDTA blood samples
2.3 Procedures and principles
2.3.1 Antithrombin (ATIII) assay
2.3.2 Protein C (PC) assay
2.3.3 Protein S (PS) assay
2.3.4 Screening test for lupus anticoagulants (LA1)
2.3.5 Fibrinogen assay
2.3.6 Von Willebrand factor (vWF) assay
2.3.7 Activated Partial Thromboplastin Time (APTT)
2.3.8 Complete blood count (CBC)
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Chapter III
3.0 Results
3.1 Haematological tests results
3.2 Haemostatic variables results
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Chapter 1V
4.0 Discussion
4.1 Recommendation
56
58
Chapter V
Refrences
59
Tables:
Table 1.1: Substances produced by endothelial cells
16
Table 1.2: Rate of venous thromboembolic events by age
26
Table 1.3: Rate of venous thromboembolic events by race
27
Table 1.4: Incidence of DVT in antepartum and postpartum
27
Table 1.5 Epidemiology of natural anticoagulants deficiencies
28
3
Table 1.6 Acquired Causes of natural anticoagulants deficiencies
30
Table 1.7: Levels of Natural anticoagulants of patients with (CVAs)
and controls.
30
Table 3.1: Percentage of pregnant women with low and very low
Haematological parameters
49
Table 3.2: Haematological parameters for controls and pregnant
50
Table 3.3: Haemostatic variables of controls and pregnant women
52
Table 3.4: Levels of coagulation cascade proteins and coagulation
tests during pregnancy and puerperium
54
Figures:
Figure 1.1 Haemostasis: a system in balance
13
Figure 1.2 Coagulation system: Intrinsic, Extrinsic and common
20
Figure 1.3 Action of natural inhibitors on coagulation system
21
Figure 1.4 The Fibrinolytic system
22
Figure 1.5 Physiologic balance of haemostasis
24
Figure 1.6 Prothrombotic changes associated with pregnancy
31
Figure 3.1 Haematological results for controls and pregnant women
51
Figure 3.2 Natural anticoagulants (Controls v Pregnant women)
53
Figure 3.3 Natural anticoagulants (Controls, during pregnancy and
Puerperium)
55
4
De d i c a t i o n
To all Sudanese: fathers, mothers and
their newborns.
To my wife, daughters and sons.
5
ACKNOWLEDGEMENT
I'm grateful to my supervisor: Professor Eltahir Awad Gasim Khalil, Director,
Institute of Endemic Diseases and Head, Department of Immunology and Clinical
Pathology, Institute of Endemic Diseases, University of Khartoum, who has helped
me so much to set and complete this research and to format it in this way. I can't
adequately acknowledge his hard work. Without his supervision this thesis could
never have been completed.
I wish to thank my co- supervisor: Professor Merghani Ali Mohammed Ahmad,
consultant hematologist, King Faisal University, Saudi Arabia, who gave me
extremely helpful suggestions in this viable academic discipline.
My deep and cordial thanks are also extended to Mr. Abdulgader Haj Al-Agib, Mr.
Adel Naser and Mrs. Fathia Adam for their great help and advice to finish this work.
Thanks are also due to Dr. Abdulrahman M.A. Tambal. Associate Prof. Senior
Consultant hematologist, Rabat University, Sudan; and Dr. Ahmad M. Musa. Institute
of Endemic Diseases, U.of.K & Head of Leishmaniasis East Africa Platform.
Finally, I would like to express my gratitude to my family.
6
‫ﻤﻠﺨﺹ ﺍﻟﺩﺭﺍﺴﺔ‪:‬‬
‫ﺨﻠﻔﻴﺔ‪:‬‬
‫ﻤﻀﺎﻋﻔﺎﺕ ﺍﻟﺤﻤل ﺘﺘﺴﺒﺏ ﻓﻲ ﻤﻭﺕ ﺤﻭﺍﻟﻲ ‪ 600,000‬ﻤﻥ ﺍﻟﺤﻭﺍﻤل ﺴﻨﻭﻴﺎ ﻓﻲ ﺍﻟﻌﺎﻟﻡ‪ .‬ﻨﺴﺒﺔ ﻋﺎﻟﻴﺔ ﺠﺩﺍ ﻤﻥ‬
‫ﻫﺫﺍ ﺍﻟﻌﺩﺩ‬
‫) ‪ ( %98‬ﺘﺤﺩﺙ ﺒﺎﻷﻗﻁﺎﺭ ﺍﻟﻨﺎﻤﻴﺔ ﻭ ﺨﺎﺼﺔ ﺍﻟﻤﺠﺘﻤﻌﺎﺕ ﺍﻟﻔﻘﻴﺭﺓ ﻤﻨﻬﺎ‪ .‬ﺘﻌﺘﺒﺭ ﻤﻀﺎﻋﻔﺎﺕ ﺍﻟﺤﻤل‬
‫ﺍﻟﺴﺒﺏ ﺍﻟﺭﺌﻴﺱ ﻟﻤﻭﺕ ﺍﻟﻨﺴﺎﺀ ﺃﺜﻨﺎﺀ ﻓﺘﺭﺓ ﺍﻟﻌﻤﺭ ﺍﻻﻨﺠﺎﺒﻰ ﻓﻲ ﺍﻷﻗﻁﺎﺭ ﺍﻟﻔﻘﻴﺭﺓ‪.‬‬
‫ﺃﺜﻨﺎﺀ ﺍﻟﺤﻤل ﻭ ﺒﻌﺩ ﺍﻟﻭﻻﺩﺓ ‪ ،‬ﻫﻨﺎﻟﻙ ﺘﻐﻴﺭﺍﺕ ﻁﺒﻴﻌﻴﺔ ﺘﺤﺩﺙ ﻓﻲ ﺃﺠﻬﺯﺓ ﺤﻔﻅ ﺴﻴﻭﻟﺔ ﺍﻟﺩﻡ ﺒﺎﻟﻤﻌﺩل ﺍﻟﻁﺒﻴﻌﻲ ‪ ،‬ﻭ‬
‫ﺫﻟﻙ ﻗﺩ ﻴﺴﺒﺏ ﺘﺨﺜﺭ ﺍﻟﺩﻡ ﺃﻭ ﺤﺩﻭﺙ ﺍﻟﻨﺯﻑ ‪ .‬ﻴﻜﻭﻥ ﻤﻌﺩل ﺍﻟﺘﻐﻴﺭ ﻓﻲ ﺍﺘﺠﺎﻩ ﺍﻟﺘﺨﺜﺭ ﺃﻜﺜﺭ ﻤﻨﻪ ﻟﻠﺴﻴﻭﻟﺔ ﺒﻌﺩ‬
‫ﺍﻟﺸﻬﺭ ﺍﻟﺜﺎﻟﺙ ﻟﻠﺤﻤل ‪ .‬ﻫﺫﻩ ﺍﻟﺨﺎﺼﻴﺔ ﻟﺤﻤﺎﻴﺔ ﺍﻷﻡ ﻤﻥ ﺃﻱ ﻨﺯﻑ ﻤﺤﺘﻤل ﺃﺜﻨﺎﺀ ﺍﻟﻭﻻﺩﺓ ﺍﻟﻁﺒﻴﻌﻴﺔ ‪ ،‬ﻭ ﻟﻜﻨﻬﺎ ﻗﺩ‬
‫ﺘﻌﺭﺽ ﺍﻷﻡ ﺃﻭ ﺍﻟﻁﻔل ﺇﻟﻰ ﻤﻀﺎﻋﻔﺎﺕ ﺃﺨﺭﻯ ﻤﺜل ﺍﻟﺠﻠﻁﺎﺕ ‪ ،‬ﺃﻤﺭﺍﺽ ﺍﻟﻤﺸﻴﻤﺔ ‪ ،‬ﺍﻹﺠﻬﺎﺽ ﺍﻟﻤﺘﻜﺭﺭ ‪ ،‬ﻨﻘﺹ‬
‫ﻭﺯﻥ ﺍﻟﻁﻔل ‪ ،‬ﺃﻭ ﺘﺴﻤﻡ ﺍﻟﺩﻡ ﺨﺎﺼﺔ ﻓﻲ ﻭﺠﻭﺩ ﻋﻭﺍﻤل ﺃﻀﺎﻓﻴﺔ ‪.‬‬
‫ﺍﻷﻫﺩﺍﻑ‪- :‬‬
‫‪ .1‬ﻗﻴﺎﺱ ﻨﺴﺒﺔ ﺍﻟﻤﺜﺒﻁﺎﺕ ﺍﻟﻁﺒﻴﻌﻴﺔ ﻟﺘﺨﺜﺭ ﺍﻟﺩﻡ ) ﻤﻀﺎﺩ ﺍﻟﺜﺭﻭﻤﻴﻥ ‪ ،‬ﺒﺭﻭﺘﻴﻥ ﺴﻰ‪ ،‬ﺒﺭﻭﺘﻴﻥ ﺍﺱ( ﻓﻲ ﺍﻟﺤﻭﺍﻤل‬
‫ﺍﻟﺴﻭﺩﺍﻨﻴﺎﺕ ﻭ ﻤﺘﺎﺒﻌﺘﻬﻥ ﺒﻌﺩ ﺍﻟﻭﻻﺩﺓ ﻷﻱ ﺘﻐﻴﺭﺍﺕ ﻤﺤﺘﻤﻠﺔ ﺃﻭ ﻤﻀﺎﻋﻔﺎﺕ ﻗﺩ ﺘﺤﺩﺙ ﻟﻸﻡ ﺃﻭ ﺍﻟﻁﻔل ﻗﺒل ﺃﻭ ﺃﺜﻨﺎﺀ‬
‫ﺃﻭ ﺒﻌﺩ ﺍﻟﻭﻻﺩﺓ ‪.‬‬
‫‪ -2‬ﻤﻘﺎﺭﻨﺔ ﻫﺫﻩ ﺍﻟﻨﺘﺎﺌﺞ ﻤﻊ ﻨﺴﺒﺔ ﺍﻟﻤﺜﺒﻁﺎﺕ ﺍﻟﻁﺒﻴﻌﻴﺔ ﻓﻲ ﺍﻟﻨﺴﺎﺀ ﺍﻟﺴﻭﺩﺍﻨﻴﺎﺕ ﺍﻟﻐﻴﺭ ﺤﻭﺍﻤل‪.‬‬
‫ﺍﻟﻁﺭﻴﻘﺔ‪-:‬‬
‫ﻫﺫﻩ ﺩﺭﺍﺴﺔ ﻤﻘﺎﺭﻨﺔ )‪ ( 3 :1‬ﺃﺠﺭﻴﺕ ﺒﺎﻟﻤﻤﻠﻜﺔ ﺍﻟﻌﺭﺒﻴﺔ ﺍﻟﺴﻌﻭﺩﻴﺔ‪ ،‬ﺠﺎﻤﻌﺔ ﺍﻟﻤﻠﻙ‬
‫ﻓﻴﺼل‪،‬‬
‫ﺸﻤﻠﺕ ‪ 40‬ﻤﻥ ﺍﻟﻨﺴﺎﺀ ﺍﻟﺴﻭﺩﺍﻨﻴﺎﺕ ﺍﻟﺤﻭﺍﻤل ﺒﻌﺩ ﺍﻟﺸﻬﺭ ﺍﻟﺜﺎﻟﺙ ‪ ،‬ﺃﻋﻤﺎﺭﻫﻥ ﺒﻴﻥ ‪ 22‬ﺇﻟﻰ ‪ 38‬ﻋﺎﻡ‬
‫) ﻤﺘﻭﺴﻁ ‪ 30‬ﻋﺎﻡ(‪ .‬ﻭ‪ 120‬ﻤﻥ ﺍﻟﻨﺴﺎﺀ ﺍﻟﺴﻭﺩﺍﻨﻴﺎﺕ ﺍﻟﻐﻴﺭ ﺤﻭﺍﻤل ﺃﻋﻤﺎﺭﻫﻥ ﺒﻴﻥ ‪ 20‬ﺇﻟﻰ ‪ 38‬ﻋﺎﻡ)ﻤﺘﻭﺴﻁ ‪29‬‬
‫‪7‬‬
‫ﻋﺎﻡ(‪ .‬ﺘﻡ ﺃﺨﺫ ﻋﻴﻨﺎﺕ ﺍﻟﺩﻡ ﻤﻥ ﺍﻟﺤﻭﺍﻤل ﺃﺜﻨﺎﺀ ﻓﺘﺭﺓ ﺍﻟﺤﻤل ﺜﻡ ﺒﻌﺩ ﺍﻟﻭﻻﺩﺓ ‪ ،‬ﻜﻤﺎ ﺠﻤﻌﺕ ﺍﻟﻌﻴﻨﺎﺕ ﻤﻥ ﺍﻟﻐﻴﺭ ﺤﻭﺍﻤل‬
‫ﻓﻰ ﺃﻨﺎﺒﻴﺏ ﺴﺘﺭﺍﺕ ﺍﻟﺼﻭﺩﻴﻭﻡ ﻭ ‪. EDTA‬‬
‫ﺃﺠﺭﻴﺕ ﺘﺤﺎﻟﻴل ﻓﺤﺹ ﺍﻟﺩﻡ ﺍﻟﻜﺎﻤل )‪ , (CBC‬ﻗﻴﺎﺱ ﻤﺜﺒﻁﺎﺕ ﺘﺨﺜﺭ ﺍﻟﺩﻡ ﺒﺭﻭﺘﻴﻥ ﺴﻰ ‪ ،‬ﺒﺭﻭﺘﻴﻥ ﺍﺱ ﻭ‬
‫ﻤﻀﺎﺩ ﺍﻟﺜﺭ ﻭﻤﺒﻴﻥ ‪ .AT111‬ﻭ ﻜﺫﻟﻙ ﻗﻴﺎﺱ ﺯﻤﻥ ﺍﻟﺜﺭﻭﻤﺒﻭﺒﻼﺴﺘﻴﻥ‬
‫‪ ،APTT‬ﻤﻀﺎﺩﺍﺕ ﺍﻟﻠﻭﺒﺱ ‪، LA‬‬
‫ﺍﻟﻔﺎﻴﺒﺭﻭﻨﻭﺠﻴﻥ ‪ ، FIB‬ﻭ ﻋﺎﻤل ﻓﻭﻨﻭﻟﺒﺭﺍﺩ ‪. vWF‬‬
‫ﺍﻟﻨﺘﺎﺌﺞ‪-:‬‬
‫ﻫﻨﺎﻟﻙ ﺘﻐﻴﺭﺍﺕ ﻭﺍﻀﺤﺔ ﻓﻰ ﻨﺘﺎﺌﺞ ﻓﺤﺹ ﺍﻟﺩﻡ ﺍﻟﻜﺎﻤل ‪ ،‬ﻭ ﺫﻟﻙ ﺒﺎﻨﺨﻔﺎﺽ ﺍﻟﻬﻴﻤﻭﻗﻠﻭﺒﻴﻥ ﻭ ﺍﻟﻬﻴﻤﺎﺘﻭﻜﺭﻴﺕ ‪ .‬ﺃﻤﺎ‬
‫ﻤﺘﻭﺴﻁ ﺤﺠﻡ ﻜﺭﻴﺔ ﺍﻟﺩﻡ ﺍﻟﻭﺍﺤﺩﺓ ‪ MCV‬ﻓﻘﺩ ﺯﺍﺩ ﻓﻰ ﺒﻌﺽ ﺍﻟﺤﻭﺍﻤل ﻤﻤﺎ ﻴﺩل ﻋﻠﻰ ﻨﻘﺹ ﺍﻟﻔﻭﻟﻴﺕ ‪ ،‬ﻭ ﺍﻨﺨﻔﺽ‬
‫ﻓﻰ ﺒﻌﻀﻬﻥ ﻤﻤﺎ ﻴﺩل ﻋﻠﻰ ﻨﻘﺹ ﺍﻟﺤﺩﻴﺩ ‪ .‬ﺃﻤﺎ ﺘﻌﺩﺍﺩ ﺍﻟﺼﻔﺎﺌﺢ ﺍﻟﺩﻤﻭﻴﺔ ﻓﻘﺩ ﻜﺎﻥ ﻁﺒﻴﻌﻴﺎ ﻤﺎ ﻋﺩﺍ ﻨﺴﺒﺔ ﻗﻠﻴﻠﺔ‬
‫ﺍﻨﺨﻔﻀﺕ ﻓﻴﻬﺎ ﺍﻟﺼﻔﺎﺌﺢ ﻭ ﻤﺎ ﻟﺒﺜﺕ ﺃﻥ ﺍﺭﺘﻔﻌﺕ ﻟﻠﻤﻌﺩل ﺍﻟﻁﺒﻴﻌﻲ ﺒﻌﺩ ﺍﻟﻭﻻﺩﺓ )‪.(PIT‬‬
‫ﻟﻡ ﺘﺘﻐﻴﺭ ﻨﺘﺎﺌﺞ ‪ (P =0.9) APTT‬ﻭ ﻤﻀﺎﺩﺍﺕ ﺍﻟﻠﻭﺒﺱ‪.(P =0.2)) .‬‬
‫ﻨﺴﺒﺔ ﺘﺭﻜﻴﺯ ﺒﺭﻭﺘﻴﻥ ‪S‬‬
‫)‪(P =0.5‬‬
‫ﻭ‬
‫ﺍﻨﺨﻔﻀﺕ ﻜﺜﻴﺭﺍ ﺃﺜﻨﺎﺀ ﺍﻟﺤﻤل )‪ ، (P = 0.001‬ﺒﻴﻨﻤﺎ ﻟﻡ ﻴﺘﻐﻴﺭ ﺘﺭﻜﻴﺯ ﺒﺭﻭﺘﻴﻥ ‪C‬‬
‫‪. (P‬‬
‫ﻤﻀﺎﺩ ﺍﻟﺜﺭ ﻭﻤﺒﻴﻥ ‪=0.9) AT111‬‬
‫ﺃﻤﺎ ﻋﺎﻤل ﺍﻟﻔﻭﻨﻭﻟﺒﺭﺍﻨﺩ ‪ (P =0.01) Vwf‬ﻭ‬
‫ﺍﻟﻔﺎﻴﺒﺭﻨﻭﺠﻴﻥ )‪ (P =0.001‬ﻓﻘﺩ ﺍﺭﺘﻔﻌﺎ ﻜﺜﻴﺭﺍ ﺃﺜﻨﺎﺀ ﺍﻟﺤﻤل‪.‬‬
‫ﻜل ﻨﺘﺎﺌﺞ ﺍﻟﺘﺤﺎﻟﻴل ﺍﻟﺴﺎﺒﻘﺔ ﻋﺎﺩﺕ ﺇﻟﻰ ﻤﻌﺩﻻﺘﻬﺎ ﺍﻟﻁﺒﻴﻌﻴﺔ ﺒﻌﺩ ﺍﻟﻭﻻﺩﺓ ﻭ ﺃﺼﺒﺤﺕ ﻤﺘﺠﺎﻨﺴﺔ ﺘﻤﺎﻤﺎ ﻤﻊ ﻨﺘﺎﺌﺞ‬
‫ﻤﺠﻤﻭﻋﺔ ﺍﻟﻨﺴﺎﺀ ﺍﻟﻐﻴﺭ ﺤﻭﺍﻤل‪.‬‬
‫ﺍﻟﺨﻼﺼﺔ‪:‬‬
‫ﻴﺼﻴﺏ ﺍﻟﺤﻤل ﻓﻲ ﺍﻟﻨﺴﺎﺀ ﺍﻟﺴﻭﺩﺍﻨﻴﺎﺕ ﺃﺠﻬﺯﺓ ﻨﺨﺜﺭ ﻭ ﺴﻴﻭﻟﺔ ﺍﻟﺩﻡ ﺒﺎﺘﺨﻔﺎﺽ ﻭﺍﻀﺢ ﻓﻲ ﺒﺭﻭﺘﻴﻥ ﺍﺱ ﻤﻊ ﻋﺩﻡ‬
‫ﺘﻐﻴﺭ ﻤﻀﺎﺩ ﺍﻟﺜﺭ ﻭﻤﺒﻴﻥ ﺃﻭ ﺒﺭﻭﺘﻴﻥ ‪ C‬ﻤﻤﺎ ﻴﻌﺭﻀﻬﻥ ﻟﻤﻀﺎﻋﻔﺎﺕ ﺍﻟﺤﻤل‪.‬‬
‫‪8‬‬
9
Abstract:
Background:
Six hundred thousands women die annually of pregnancy-related causes. The majority
(98%) of these deaths occurs in developing countries. Pregnancy complications are
the leading causes of death among women of reproductive age in developing
countries.
During pregnancy and puerperium, many changes affect the coagulation and
fibrinolytic systems predisposing pregnant women to thrombotic episodes. The
overall balance shifts towards hypercoagulability. This phenomenon is probably due
to hormonal changes to protect pregnant women from fatal haemorrhage during
delivery, but predispose them to thromboembolic phenomena especially if one or
more additional congenital or acquired risk factor(s) ensue.
Objective:
To determine possible changes in the levels of blood natural anticoagulants [AT111,
PC and PS] in pregnant and non-pregnant Sudanese women, and to follow up them
after delivery.
Materials & Methods:
This was a prospective case control study (1:3) that was carried out at King Faisal
University Teaching Hospital, Saudi Arabia. Following informed consent, forty
pregnant Sudanese women (n= 40) with no aberrant disease. One hundred and twenty
non-pregnant Sudanese women attending the same hospital for routine gynecological
follow up were also enrolled.
10
Blood samples were collected in trisodium citrate for coagulation studies and EDTA
for CBC. The coagulation variables performed are AT111, PC, PS, Fib, vWF, APTT,
and LA1.
Results:
The haemostatic variables APTT (p=0.9), LA1 (p=0.2) were found to be comparable
between the two groups. Fibrinogen levels (p=0.001), and von Willibrand factor
(p=0.01), were significantly elevated in the pregnant women compared to controls.
Protein S was significantly reduced in the pregnant women (p=0.001), while protein
C and ATIII remained unchanged throughout pregnancy (p=0.5 & p=0.9
respectively). During the puerperium, fibrinogen, von Willibrand factor and protein S
levels of pregnant women returned to levels that were not significantly different from
those of the controls.
Conclusion:
Coagulopathic derangements of pregnancy in Sudanese women, is mainly affecting
fibrinogen and protein S levels, with no significant change in protein C and ATIII
levels. The increase probably predisposes pregnant women to increased thrombotic
episodes that are characteristic of some pregnancies. The role of other factors, like
prothrombin gene mutation and Methylene-tetrahydro-folate reductase deficiency
needs further investigations in these women.
11
LIST OF ABBREVIATIONS:
APCR
Activated protein C resistant.
APTT
Activated Partial Tromboplastin Time
ATIII
Antithrombin III
CBC
Complete Blood Count
DVT
Deep venous thromboembolism
EDTA
Ethylenediaminetetra-aectic acid
Fib
Fibrinogen
FVL
Factor V Leiden (FVL)
LA
Lupus Anticoagulant
MPD
Myloproliferative disorders
MTHFR
Mehtylenetetrahydrofolate reductase deficiency
PC
Protein C
PIT
Pregnancy-induced thrombocytopenia
PLT
Platelet
PS
Protein S
vWF
von Willebrand factor
12
Objective:
- To determine the levels of blood natural anticoagulants in pregnant Sudanese women
living in Saudi Arabia (eastern province) as possible risk factors for thrombotic
episodes (thrombophilia).
Rationale:
Risk factor for venous thromboembolism and pregnancy complications in expatriate
Sudanese women were not studied before. The rate of venous thromboembolism in
black pregnant women is 2.64 per 1000 deliveries, Table 1.3. The collected data will
help in planning programs for preventing or minimizing of DVT in high risk situations
such as surgery or immobilization during and after pregnancy. The study will help to
reduce pregnancy-associated maternal and fetus complications such as miscarriage,
intrauterine growth restriction and pre-eclampsia. Also it will help to set appropriate
screening and treatment protocols for pregnant women with predisposing risk factors
for pregnancy-associated thrombosis.
Hypothesis:
Venous thrombo-embolism and pregnancy-associated complications are caused by
defective maternal haemostasis due to physiological changes of natural anticoagulants
or presence of Lupus anti-coagulant antibodies.
13
Chapter 1
1.0 Introduction and literature review:
1.1 Definition:
Haemostasis is a protective process by which the body stops flowing following injury
as well as maintaining the blood in a fluid state within the vascular compartment. i.e.
bleeding-clotting balance.1 Fig: 1.1
Fig 1.1. Haemostasis: a system in balance.
14
Five major systems are involved in maintaining haemostasis:
1. Vascular endothelium 2. Platelets 3. Coagulation cascade 4. Natural anticoagulants.
5. Fibrinolytic system.2
1.1.1 Vascular endothelium (Endothelial system):
Several processes are exerted by the vascular endothelium to prevent bleeding:
1. Contraction of blood vessels (vasoconstriction) 2. Diversion of blood flow around
damaged vasculature 3. Initiation of contact-activation of platelets with subsequent
aggregation 4. Contact-activation of the coagulation systems (both extrinsic and
intrinsic) leading to fibrin formation. Figure: 1.2.
The blood vessel intimal surface is covered with endothelial cells (ECs). The ECs
play a key role in the body's defense response, by possessing surface receptors for a
variety of physiological substances, such as thrombin. Following trauma or
stimulation by high shear stress, thrombin/cytokines will be expressed on its surface
and released into the plasma. The healthy vascular endothelium possesses a wide
range of anti-thrombotic properties. These result in the neutralization of thrombin,
lysis of fibrin and inhibition of platelets/sub endothelial and platelets/platelets
interactions.3, 4 Table 1.1
a] Endothelial cell activities affecting platelets–vessel wall interaction:
Normally, the blood vessels’ endothelial lining is smooth with a negatively charged
layer. When the vessel wall is injured, the lining loses its negative charge and
becomes rough leading to platelets aggregation and adhesion as well as triggering the
coagulation cascade.
15
Prostaglandins and nitric oxide produced by ECs act on smooth muscle cells of the
vessel wall and help to modulate blood flow. Both substances inhibit aggregation of
platelets and leucocytes.5 The von Willebrand factor (vWF) synthesized in ECs and
megakaryocytes influences platelet-vessel wall interaction. The ECs contain
adenosine diphosphate (ADP) which is important for inducing platelet-aggregation.6, 7
b] Endothelial pro-coagulant activity:
Tissue factor (TF) expressed after ECs are activated by endotoxins or by leucocytes
after stimulation by cytokines, leads to rapid localized thrombin generation. The
natural inhibitor of TF known as tissue factor pathway inhibitor (TFPI) is also
synthesized and secreted from ECs.8
c] Endothelial cell–derived coagulation inhibitors:
Thrombomodulin is an anticoagulant protein which is synthesized and expressed on
ECs. It changes the function of thrombin from pro-coagulant to anticoagulant.9
d] Endothelial cell–derived fibrinolytic factors:
The fibrinolytic factors, tissue plasminogen activator (tPA), and plasminogen
activator inhibitor type 1(PA I – 1) are synthesized primarily by ECs, while urinary
plasminogen activator (urokinase) is mainly from the kidney and the gut.10
16
Table1.1 Anti-coagulant substances of endothelial cells
Factors secreted by the endothelium
Activities
Prostacylin
Vasodilation inhibits platelets aggregation.
Nitric oxide
Vasodilation
inhibits
platelets
adhesion,
aggregation, leukocytes adhesion and smooth
muscle hyperplasia following vascular injury.
Tissue plasminogen activator (tPA)
Regulates fibrinolysis.
Thrombomodulin
Anticoagulant activity
Thromboplastin
Promotes blood coagulation
Platelet activating factor
Activation of platelets and neutrophils.
Promotes platelets adhesion and activation of
von Willebrand Factor
blood coagulation.
17
1.1.2 Platelets System :( Platelets plug formation):
The temporary platelets plug is a temporary loose platelets plug formed by platelets
adhesion, shape change, release reaction, aggregation, pro-coagulant activities and
platelets contact factors (primary haemostasis). Platelets escaping from an injured
blood vessel adhere to vessel wall particularly to collagen. Adhesion is a reversible
process whereby platelets stick to foreign surfaces. Then they undergo a change in
shape, becoming more spherical and putting out long, spiny pseudopods.11 A variety
of soluble substances, including ADP, vWF and fibronectin could operate in a similar
way.12
The vWF which is synthesized in ECs and megakaryocytes influences platelets-vessel
wall interaction. Platelet’s vWF is stored in α granules, and released into the plasma
during platelets activation. In plasma, vWF is found closely associated with
coagulation factor VIII. The complex protects factor VIII from proteolysis by protein
C. Failure of vWF to bind factor VIII, leads to its rapid turnover and to
FVIII
deficiency.13
1.1.3 Coagulation System:
The stage involved in arresting bleeding after vessel damage is the formation of a
stable platelet plug. This stabilization is achieved through the formation and
deposition of fibrin clot, the end product of coagulation (secondary haemostasis).
The coagulation proteins are produced in the liver and can be categorized into three
groups: substrates, co-factors, and enzymes. The main substrate of the blood
coagulation system is factor 1 (fibrinogen) from which a fibrin clot is formed.
Co-factors are proteins that accelerate the enzymatic reactions involved in the
coagulation process, such as factors III, V, VIII. The enzymes involved in coagulation
18
can be subdivided into two groups: serine proteases or transaminases. Except for
factor XIII (fibrin-stabilizing factor), all the enzymes are serine proteases when they
are in their activated form. The formation of a fibrin clot involves a series of
biochemical reactions. This process requires plasma proteins (coagulation factors) as
well as phospholipids and calcium. Blood coagulation leading to fibrin formation can
be divided arbitrary into two pathways: intrinsic and extrinsic. Both of which share
specific coagulation factors with the common pathway. Both pathways require
initiation, which leads to subsequent activation of various coagulation factors.14 Fig
1.2.
1.1.4 Natural anticoagulants system:
The blood coagulation process can be activated rapidly when the need arises. This
involves the generation of proteolytic enzymes, such as thrombin which are
potentially lethal if their action is not limited. Physiological anticoagulants fall into
two main groups: 15 Fig 1.3.
A] Anti-thrombins: these inhibit the serine proteases of coagulation cascade.
B] Components of protein C system, which neutralize activated coagulation factors.
In addition to the specific inhibitors, there are some other inhibitory mechanisms. One
of these is the detoxifying property of the liver, which plays an important role in
removal of activated clotting factors, directly or after their combination with natural
inhibitors. Furthermore, the free thrombin can be removed as a result of its adsorption
on to fibrin degradation products.16
19
1.1.5 Fibrinolytic System (fibrin- lysing):
Fibrinolysis is the physiologic process of removing unwanted fibrin deposits. It is
mediated mainly by the enzyme plasmin generated from plasminogen which acts
primarily on fibrin to produce soluble fragments, which are removed by the
reticuloendothelial system. In addition to plasmin, plasminogen, and plasminogen
activators, inhibitors of plasmin are a part of the fibrinolytic system which keep
fibrinolysis from getting out of control.17 Fig 1.4
20
Intrinsic Pathway
Prekal
likrein
Kallikrein
XIIa
XII
Extrinsic Pathway
XIa
XI
III
VII
Ca++
IXa
IX
VII
Ca++
VIII
PF3
Xa
X
Common Pathway
Ca++
FV
PF3
Prothrombin (II)
XIII
Thrombin (IIa)
XIIIa
Fibrinogen
Soluble
Fibrin
Monom
Figure 1.2. Coagulation System: intrinsic, Extrinsic and common pathways
21
Stable
Fibrin
Polymer
Kallikrein
Prekalli
krein
XIIa
XII
XIa
XI
Antithrombin
IXa
IX
III
VII
Ca++
III
Ca++
VIII
PF3
Protein C
Protein S
Xa
X
Ca++
V
PF3
IIa
II
Fibrinogen
Fibrin
Figure 1.3 Action of natural inhibitors of coagulation system
22
Intrinsic Activation
Exogenous Activation
Extrinsic Activation
Plasminogen
Inhibitors (Antiactivators)
Plasmin
Inhibitors (Antiplasmins)
Fibrinogen/ Fibrin
Fibrinogen degradation products (FDPs)
R.E.S
Figure 1.4 Fibrinolytic system
23
Plasminogen is activated by two different mechanisms:
A] Tissue plasminogen activator (tPA) which is synthesized by endothelial cells,
not by the liver or kidney.
B] Urinary plasminogen activator (UPA) is so called because it was first extracted
from urine. It is synthesized mainly by tubules and collecting ducts in the kidney.
Many types of body cells including endothelial cells, possess a receptor to UPA.
The plasmin can degrade completely all fibrinogen in the body within a very short
period of time. It is prevented by a number of circulating inhibitors of plasmin
(antiplasmin and plasminogen activator inhibitors).
The most potent plasmin inhibitor is α2 antiplasmin which is synthesized by the liver.
The plasmin attacks the fibrinogen forming fibrinogen degradation products (FDPs).18
figure 1.4.
24
Platelets / coagulation factors
Plasmin activation
Coagulation
Fibrinolysis
Inhibitors / Regulators
Inhibitors / Regulators
AT – 111
PAI – 1
Protein C
alpha 2 –Antiplasmin
Protein S
HRGP
HC 11
Figure 1.5 Physiologic balance of haemostasis
AT-111 = antithrombin 111; HC 11 = heparin cofactor 11; PAI = Plasminogen
activator inhibitor-1; HRGP = histidine-rich glycoprotein.
25
Disturbances of this fine balance can be responsible for several coagulation
abnormalities. A reduction in coagulability results in excessive bleeding, whereas an
increase in coagulability leads to thrombophilia.19 Fig 1.5.
Thrombophilia refers to disorders which are associated with a persistent
hypercoagulability state and increased tendency towards thrombosis.20
1.2 Classification of thrombophilia:
Thrombophilia may be inherited, acquired, or combined (multifactorial) 21, 22
1.2.1 Congenital thrombophilia:
The main congenital thrombophilias are:
Protein C deficiency (PC).
Protein S deficiency (PS).
Antithrombin III deficiency (ATIII).
Presence of factor V Leiden.
Prothrombin 20210 gene mutation.
Methylenetetrahydrofolate reductase (MTHFR) deficiency.
1.2.2 Acquired thrombophilia:
These include:
Antiphospholipids antibodies.
Myeloproliferative disorders (MPD).
Paroxysmal Nocturnal Haemoglobinuria (PNH).
Hyperhomocysteinaemia.
26
1.3 Epidemiology of maternal venous thromboembolism & natural
anticoagulants:
The clinical thrombosis is now considered a multicausal disease, resulting from
interaction between congenital and acquired risk factors. The annual incidence of
diagnosed venous thromboembolism is 1 to 2 events per 1000 of the general
population. The incidence varies widely but it is a leading cause of maternal
morbidity. The risk of maternal venous thromboembolism and pregnancy
complications for women with thrombophilia, depends on the underlying
thrombophilic defect(s), history of thrombotic events, and additional risk factors such
as operative delivery, maternal age, obesity and immobilization.23 Antenatal Deep
Vein Thrombosis (DVT) is about 0.6 per 1000 pregnancies in women younger than 35
years, and 1.7 per 1000 in those older than 35 years. The incidence rate of DVT
increases with age and race. Table 1.2 & Table 1.3 respectively. 24, 25
Table 1.2 Rate of venous thromboembolic events by age
Age
No. of cases Per 1000 deliveries 95% CI
<20
1399
1.47
(1.33-1.61)
20-24 3201
1.58
(1.50-1.66)
25-29 3667
1.67
(1.59-1.75)
30-34 3424
1.73
(1.63-1.83)
35-39 2067
2.13
(1.97-2.29)
40+
2.75
(2.36-3.14)
577
27
Table 1.3 Rate of venous thromboembolic events by race
Race
No. of cases Per 1000 deliveries 95% CI
White
5943
1.75
(1.67-1.83)
Black
2184
2.64
(2.46-2.82)
Hispanic 1699
1.25
(1.13-1.37)
Asian
266
1.07
(.85-1.29)
Other
442
1.47
(1.27-1.67)
The risk of DVT during pregnancy among women with thrombophilia in the absence
of anticoagulant therapy is about 60% for those with antithrombin III deficiency. The
risks for protein C and protein S abnormalities are lower than those for antithrombin
III. Postpartum thromboses are more common than antepartum ones.Tabe 1.4 26, 27
Table 1.4 Incidence of DVT in ante partum and postpartum for patients with
PC&PS deficiencies and Hereditary Thrombophilia (FVL & FII)
Natural anti-coagulant
Ante-partum
Post-partum
Protein C deficiency
3 - 10 %
7 - 19 %
Protein S deficiency
0 - 6 %
7 - 22 %
7.1 %
11.5 %
2.1 %
4.2 %
Hereditary Thrombophilia
Combined FVL & FII
F II only
28
Activated protein C resistance (APCR) was reported in up to 78 % of women with
DVT during pregnancy, whereas factor V Leiden was found in up to 46 %.28 Preeclampsia seems to increase the thrombotic tendency of pregnancy. In a cohort study
involving 101 women, it was found that 15% had APCR, 25% PS deficiency, 18%
hyperhomocysteinaemia, 30% anticardiolipin, and 18% had factor V Leiden. 29
Other pregnancy-associated conditions are: a) placental abruption, b) intrauterine
death, c) small-for-gestational age infant, were also assessed for thrombotic
tendencies. The study showed that 65%, 56%, and 85%, respectively, had underlying
thrombophilic disorders. 30
The deficiencies of natural anticoagulants ATIII, PC and PS
depend on the
underlying causes. Table 1-3. 31, 32, 33
Table 1.5. Epidemiology of natural anticoagulants deficiencies
natural
General
D V T
anticoagulant
population
patients
ATIII
0.02 - 1.1 %
5%
PC
0.2 %
7%
PS
0.7 - 2.3%
6%
29
1.4 The etiology of maternal thrombosis & causes of natural anticoagulants
deficiencies:
During pregnancy and puerperium, many changes affect the coagulation and
fibrinolytic systems predisposing pregnant women to thrombotic episodes.
34, 35
Virchow's classic triad for VTE, namely, hypercoagulability, venous stasis and
vascular damage, all occur in the course of uncomplicated pregnancy and delivery
These changes include: increase in clotting factors VIII, V, I and vWF, a decrease in
protein S levels and impaired fibrinolytic activity through increases in plasmonogen
activator inhibitors I and II, the later being produced by the placenta.36 As gestation
progresses, there is a significant fall in the activity of activated protein C, an
important anticoagulant factor (APCR).37 The overall balance shifts towards
hypercoagulability. This phenomenon, is probably due to hormonal changes to protect
pregnant women from fatal haemorrhage during delivery, but predispose them to
thromboembolism specially if one or more additional congenital risk factors are
present.38, 39, 40 Fig 1.6.
The major risk factors for venous thromboembolism (VTE) and pregnancy
complications include hypercoagulability of pregnancy, venous stasis, vascular
damage, bed rest for obstetric complications, operative delivery, emergency cesarean
section, advanced maternal ages, previous thromboembolism, and obesity (weight
over 80 kg).41
pathogenesis
Thrombophilias
of
miscarriage,
(congenital & acquired) may play
intrauterine
growth
restriction
part in the
and
pre-
eclampsia.42,43,44,45
Three forces can cause venous stasis: a] decreased velocity of blood flow through the
vessels which is usually caused by immobility or paralysis, b] venous dilation and
pooling, and c] venous obstruction. Venous stasis alone may not be a sufficient
30
stimulus for local thrombin generation. Under experimental conditions, blood within
an isolated vein segment remains fluid for some time. The addition of activated
factors rapidly results in thrombosis. In vivo, these activated factors are generated at
sites of tissue trauma, including operative trauma, and may then circulate and result in
fibrin generation in areas of stasis. 46Causes of natural anticoagulants (ATIII, PC and
PS) deficiencies may be inherited or acquired. 47, 48 Table 1.6. A recent study from
Sudan showed that young patients (<50 years) with Cerebro-vascular accidents
(CVAs) have a lower normal levels of Proteins C, S and ATIII. Table 3.5. Haj AlAgib, A. (2005).
Table 1-6. Acquired Causes of natural anticoagulants deficiencies.
Natural
Anticoagulant
ATIII
Acquired Causes:
Oral contraceptives, liver disease, DIC and nephritic therapy.
Oral anticoagulants, vitamin K deficiency, liver disease, DIC,
PC
newborn infants, after plasma exchange and postoperative state.
Pregnancy, oral anticoagulants, oral contraceptives, vitamin K
PS
deficiency, liver disease, diabetes type 1, acute inflammation.
Table 1.7 Levels of natural anticoagulants of patients with (CVAs) and controls.
Natural anticoagulant
Patients with (CVAs)
Controls
Protein C
60 ± 4%
122.5 ±3 %
Protein S
61±4 %
125±10 %
ATIII
55±2 %
90±5 %
31
HYPERCOAGULABILITY
Increased von Willibrand factor, Factor V, Factor VIII and Fibrinogen
Decreased Protein S and plasminogen activator inhibitor
Increased resistance to activated protein C
Change in
Blood
Components
Reduced
Blood
Flow
Vessel
Damage
Virchow's Triad
ENDOTHELIAL DAMAGE
VENOUS STASIS
Increased venous distensibility
and decreased venous tone
Vascular damage at delivery
from Caesarean section or
spontaneous vaginal delivery
50% decrease in venous flow in
lower extremity by third trimester
Enlarging uterus impedes venous
return
Figure 1.6 Prothrombotic changes associated with pregnancy
32
1.4.1 Antithrombin III (AT III) deficiency:
Antithrombin III (ATIII) an α-2 glycoprotein synthesized principally in the liver. Also
termed heparin cofactor or factor Xa inhibitor. It is the major inactivator of thrombin
and Xa. It is considered as the most important natural anticoagulant protein. The
primary enzymes which are inhibited by ATIII are FXa, FIXa and thrombin (FIIa).
Also it has inhibitory actions on FXIIa, FXIa, and complex of FVIIa and tissue
factor.49
Inactive ATIII is a slow progressive inhibitor of activated coagulation factors.
However, in the presence of heparin it becomes a very potent inhibitor of coagulation.
Heparin and heparin sulfates increase the activity of antithrombin at least 1000 fold.50
Therefore, the efficacy of heparin therapy depends on the level of ATIII.
Deficiency of ATIII is seen in approximately 2% of patients with venous
thromboembolic disease. There are two types of antithrombin III deficiency: Type I
and type II. Type I is characterized by inadequate amount of normal
antithrombin
III to inactivate the coagulation factors i.e quantitative deficiency. In type II, the
amount of antithrombin III is normal but it does not function properly i.e. qualitative
deficiency.51 Inheritance of antithrombin III deficiency is an autosomal dominant trait.
Occasionally other medical conditions, such as kidney disease (eg. nephrotic
syndrome), can cause low levels of antithrombin III. The clinical manifestations of
antithrombin III deficiency include deep venous thrombosis and pulmonary
embolism. Thrombosis may occur spontaneously or in association with
trauma, oral contraceptive pills and pregnancy.52,
53
surgery
Although ATIII level remains
normal throughout pregnancy; it is usually reduced in pre-eclampsia. The degree of
reduction of ATIII and platelet count correlate well with the severity of preeclampsia. The ATIII is a useful predictive marker for this condition.54
33
1.4.2 Protein C (PC) deficiency:
Protein C was discovered in 1960. It is a vitamin K-dependent serine protease,
synthesized in the liver and functions to inactivate factor Va and F-VIIIa. For PC to
be activated, thrombin must first bind to thrombomodulin protein, which is present on
the endothelial cell surface. Once the thrombin–thrombomodulin complex is formed,
thrombin loses its ability to convert fibrinogen to fibrin or to activate platelets, but can
convert protein C to its activated form (APC).55 APC then combines protein S(PS)
and selectively degrades FVa and
FVIIIa to limit thrombin generation, fibrin
formation and blood clotting in vivo.56 The generation of APC may be regulated by
its own specific inhibitor α-1 antitrypsin and plasminogen activator inhibitor-3(PAI-3)
[former name protein C inhibitor].
In patients with factor V Leiden (FVL), FVa resists the normal effects of APC, thus
the term activated protein C resistance–APCR – (congenital form). But such
resistance can be caused by disorders other than FVL, including antiphospholipid
antibody syndrome.57 However, in pregnancy, the resistance to APC, occurs in the
absence of FVL as a result of increased factor V and factor VIII levels (i.e. acquired
form). FVL is the leading cause of APCR, but it is not the only one. PC and PS
deficiency can give the same effect.58 PC deficiency is usually categorized into two
types: type I which results from an inadequate amount of normal protein C, whereas
type II is characterized by defective PC molecules.59 Congenital deficiency of Protein
C inherited as an autosomal dominant disorder and may account for up to 5-10% of
patients with early clotting problems. Acquired APCR was significantly more
common among women with recurrent early and late miscarriages (8.8 %) compared
with controls (3.3 %).60 The levels of protein C inhibitor gradually decreased from the
first to the third trimester. After delivery the levels rose to levels similar to those in
34
controls.61 During normal pregnancy, APCR usually increases. The APCR is
significantly higher in women with recurrent miscarriages at second trimester than
those with recurrent fetal loss in the first trimester.62 APCR was elevated in preeclamptic women, 31 % compared to 16.6 % of healthy pregnant women and 7.6 %
of normal controls.63 The use of oral contraceptives (OCs) is associated with an
increased resistance to the anticoagulant action of activated protein C (APC).64 This
phenomenon may explain the increased risk for venous thromboembolism in women
taking oral contraceptive pills. The plasma levels of factors 11, V11, V111, X and
fibrinogen are significantly increased during the use of OCs. Where as the plasma
concentration of FV is usually reduced.65
The combination of OCs use and FVL (hereditary APCR) increases the risk for
venous thrombosis by a 30-fold.66 Pregnant women with protein C deficiency may
have a slightly higher risk of placental problems during pregnancy. These include
having a small baby or pre-eclampsia.67
1.4.3 Protein S (PS) deficiency:
Protein S is a vitamin K–dependent protein. Unlike PC, it is synthesized by
hepatocytes, 68endothelial cells, 69 megakaryocytes, 70 human testis leydig cells, 71 and
brain. 72 It plays an important role in the regulation of blood coagulation. It serves as a
cofactor to protein C by forming a complex with APC which degrades FVa and
FVIIIa.73 Recent data showed that PS has an independent anticoagulant activity not
related to PC, but by direct inhibition of prothrombinase and tenase activities.74
Protein S circulates in human plasma into two forms, a) free form which comprises
40% b) bound form, constituting 60 % and binds to an additional protein. Only free
protein S has a cofactor activity for APC.75, 76
35
Hereditary protein S deficiency is an autosomal dominant trait. The first deficiencies
were described in 1984 by several groups.77 The prevalence of PS deficiency in the
general population varies between 0.005 and 0.7 to 2.3 %.78 It is found in up to 10 %
of patients with congenital thrombophilia.79 Hereditary PS deficiency is relatively
rare, but some medical conditions may lead to acquired PS deficiency, such as
pregnancy, oral anticoagulant therapy, oral contraceptives, liver disease, nephrotic
syndrome and disseminated intravascular coagulation.80
Free PS levels falls significantly from the first to the second trimesters, but no further
depletion occurs during the third trimesters.81 The decrease in PS levels partially
explains the acquired APCR. The reduction of PS with an increased APCR, result in a
hypercoagulable state.82 The increasing volume of plasma during normal pregnancy
and its dilution effect might play some role in low PS activity.83
A recent study by Liberti and co-workers, showed that PS levels are influenced by
gender (women have lower PS levels than men) and by age (total PS levels increase
with age in women).84 Total and free PS levels are also positively correlated with
cholesterol and triglyceride levels respectively.85
There are three types of PS deficiency: Type I that results from an inadequate amount
of PS, both free and bound forms. Type II is characterized by defective PS molecules.
Type III is characterized by decreased amount of free PS but normal total PS.86 It is
well established that the deficiency of PS was associated with recurrent fetal loss and
late non-recurrent fetal loss.87
1.4.4 Factor V Leiden (FVL), increased Activated Protein C Resistance (APCR):
FVL is the most common congenital cause of thrombophilia. It results from adenine
506 guanine (A506G) mutation in factor V (FVL) due to the substitution of arginine
36
at position 506 with glutamine. FVL is found with a high frequency (20– 60 %) in
patients with thrombosis. The natural anticoagulant PC with PS as a co-factor breaks
down the activated FV (FVa). In patients with FVL, the FVa in the blood is more
resistant to be broken down by activated PC, so the nickname Activated Protein C
Resistance (APCR). The clotting process will continue towards fibrin formation,
leading to venous thrombosis.88
The risk of venous thrombosis is about eight times greater for individuals with
heterozygous FVL and 80 times greater for individuals with homozygous compared
with those without the condition. Most individuals with FVL have a low risk of
venous thrombosis, but the risk factor will increase if one or more additional risk
factors are present such as major surgery, immobility for long period, pregnancy or
oral contraception.89
1.4.5Prothrombin20210:
The condition known as prothrombin 20210 is due to a mutation of the prothrombin
gene. It is inherited as autosomal dominant. Individuals with prothrombin 20210
mutation have low risk factor of venous thrombosis unless one or more additional risk
factors are present such as FVL, PC, PS deficiency, major surgery, reduced mobility,
use of oral contraceptives and pregnancy.90
1.4.6 Methylenetetrahydrofolate reductase deficiency (MTHFR):
Elevated level of plasma homocysteine (hyperhomocysteinemia) is a well established
risk factor for both arterial and venous thrombosis.91 Hyperhomocysteinemia may be
caused by nutritional deficiencies or by defects in the enzymes involved in
homocysteine metabolism such as methylenetetrahydrofolate reductase (MTHER).92
37
Hyperhomocysteinaemia may be a risk for pre-eclampsia, because of the toxic effects of
homocysteine on the endothelium.93 Deficiencies of vitamins B6 or B12 can lead to
acquired homocysteinaemia, which is treatable with folic acid and vitamin B6 or B12
supplements. Elevated homocysteine and reduced serum folate concentrations are risk
factors for recurrent spontaneous early pregnancy loss.94 If so; it may be useful to
measure folic acid, vitamin B6 and B12 in women with adverse pregnancy outcome.
1.4.7 Myeloproliferative disorders (MPD):
Myeloproliferative disorders (MPD) include blood disorders such as polycythaemia
rubra Vera, idiopathic myelofibrosis, chronic myelogenous leukaemia and essential or
primary thrombocythaemia. The blood viscosity is a major determinant of flow. The
main contributors to blood viscosity are the concentrations of red cells and fibrinogen,
as well as the tendency of erythrocytes to aggregate. The increased blood viscosity, and
hence disturbed flow, is a major pathogenic factor leading to thrombosis in primary
polycythemia.
Polycythaemia rubra Vera is due to a sustained elevation in the red cell mass. There
may also be an elevated platelet and white cell count as well. Individuals with this
condition have an increased risk of both arterial and venous thrombosis unless the mass
of cells is reduced with venesection or certain cytotoxic drugs.95
1.4.8 Anti-phospholipids antibodies:
Antiphospholipid syndrome (APS) results from the presence of antiphospholipid
antibodies in the blood, combined with a previous thrombosis, specific problems during
pregnancy, or both. Some women with antiphospholipid antibodies may have
miscarriage during the first trimester.96 In some pregnant women, antiphospholipid
38
antibodies can cause thrombosis in the small blood vessels of the placenta which
impairs the blood supply to the fetus leading to intra-uterine growth retardation (IUGR)
and may to foetal death.97 Some time, the damage of placenta impairs the growing of
the foetus to normal size. In other cases the damaged placenta may lead to preeclampsia.98Therefore, the indications for testing antiphospholipids are: a) recurrent
pregnancy losses. b) A history of unexplained poor fetal growth. c) Early onset of
severe pre-eclampsia. d) Unexplained placental abruption.
1.4.9 Diagnosis of venous thromboembolism:
Deep venous thrombosis may be diagnosed by several screening techniques such as
ultrasound, venography and recently magnetic resonance imaging (MRI). If the
thrombus is too small to cut off the blood flow, the signs and symptoms of DVT may be
nonspecific or absent. So the laboratory tests are necessary to predict and diagnose the
clotting abnormalities that lead to hypercoagulability.99
Ultrasonography:
Ultrasonography is the most frequently used imaging method for diagnosing DVT
because it is accurate for detecting proximal thrombus, non-invasive, and widely
available.
Venography:
Ascending contrast venography is the traditional gold standard test for diagnosing DVT,
but is rarely used in clinical practice because it is labour-intensive, requires injection of
contrast dye, and is uncomfortable for the patient. 100
39
Chapter II
2.0 Materials and Methods:
2.1 Study design:
This was a case control study (1:3) conducted in King Faisal University Teaching
Hospital, Saudi Arabia.
2.2 Patients & Samples:
The study proposal was reviewed and approved by the scientific committee, Faculty
of Medical Laboratory Sciences, U. of Khartoum, Sudan and the Hospital Committee
in King Faisal University Hospital, Saudi Arabia. Written informed consent was
obtained from each woman and her husband following through explanation about the
objectives and nature of the study. A Case Record Form (CRF) with demographic,
clinical and laboratory data was designed and filled for each woman.
2.2.1 Patients’ inclusion criteria:
Forty pregnant Sudanese women with no aberrant complications were enrolled in the
study. Volunteers have different ages and in 3rd gestation.
2.2.2 Controls’ inclusion criteria:
One hundred and twenty healthy non-pregnant Sudanese women with different ages
who report to the same hospital were also recruited as controls.
40
2.2.3 Samples:
Three blood samples were collected from each volunteer and control in a plain, trisodium citrate and EDTA anticoagulants respectively. The blood samples were
collected by evacuated tube system which offers the benefit of multisampling blood
collection. The evacuated tube system consisted of a disposable sterile needle, a needle
holder and a color-coded evacuated tube.
2.2.4 Serum preparation:
Blood samples were collected in plain tubes, incubated at 37 0C for 1-2 hours, and then
centrifuged at 3000 rpm for 10 minutes. Then the serum separated, divided into small
plastic aliquots of one ml each and frozen at -80 0C. Immediately before performing the
tests, the serum was thawed rapidly at 37 0C water-bath for 10 minutes.
2.2.5 Preparation of citrated plasma for coagulation studies:
Nine parts of whole blood were collected in one part of 3.2 or 3.8% tri-sodium citrate
anticoagulant (haematocrit value 20 to 55%). However, if the haematocrit was less than
20% or more than 55%, the amount of the anticoagulant may be adjusted using the
following formula:
C = (0.00185) (V) (100–Haematocrit). Where V = volume of blood to be collected/ml,
and C = volume of anticoagulant / ml.
Blue-topped evacuated tubes with sodium citrate had been used. The accepted blood
volume should not be less than 90% of the expected volume. Platelets-poor plasma was
obtained by centrifugation for 10 minutes at 3000 rpm immediately after blood
collection. The centrifugation of the tube with a cap on to prevent loss of CO2 which
alters the pH. The remaining materials were checked for clots. The citrated plasma was
41
separated, divided into small plastic aliquots one ml each, and frozen at -80 0C until the
analyses were performed. Immediately before testing, the plasma was thawed at 37 0C
water bath for 10 minutes. The assays were performed within 2 hours and the plasma
was not frozen again. The coagulation tests were performed according to the
manufactures package insert.
2.2.6 EDTA blood samples:
The blood samples collected in Ethylenediamine tetra-acetic acid (EDTA) anticoagulant
were tested immediately after collection, using a fully automated haematology cell
counter.
2.3 Procedures and principles:
2.3.1 Antithrombin (AT-III) assay:
Principle:
Chromogenic ATIII assays measures the functional levels of AT-III in plasma using a
substrate. Plasma containing ATIII is diluted in the presence of heparin and incubated
with excess thrombin, forming an ATIII-thrombin/heparin complex. The remaining
thrombin catalyzes the release of p-nitro-aniline (PNA) from the chromogenic substrate.
The release of PNA is measured by either an endpoint or kinetic test by measuring the
increase in absorbance at 405 nm. The absorbance obtained is increasing proportional to
the concentration of ATIII which can be quantitated from a calibration curve. The autoanalyzer used, had been calibrated by standard human plasma.
42
Procedure of ATIII measurement:
Special assay protocol for a fully-automated coagulation analyzer from Dade Behring
Company, Marburg, Germany, BCs coagulation system was used. The results were
evaluated automatically by the analyzer. The levels of control plasma, both normal and
pathological, were run with the patient's test. The reference intervals were established in
the laboratory as 80-120%.
2.3.2 Protein C (PC) assays:
Principle:
Protein C in the patient's plasma is activated by a specific snake venom activator. The
resulting activated protein C inhibits the activity of factor (Va) and factor (VIIIa). The
protein Ca is assayed in a kinetic test by measuring the increase in absorbance at 405
nm.
Procedure of (PC) measurement:
Citrated test plasma was obtained as described earlier. Special assay protocol for an
auto-analyzer from Dade Behring Company, Marburg, Germany, BCs coagulation
system was followed. Control plasma of normal and pathological ranges was run with
the test plasma. The reference range was 70-130%.
2.3.3 Protein S (PS) assays:
Principle:
Only free PS (40%) has a functional activity as a cofactor for activated PC which
inhibits (FVa) and (FVIIIa). Protein S accelerates this reaction. The coagulation time
increases proportionally to the activity of protein S in the sample. The addition of PSdeficient plasma ensures that the test mixture has a sufficient supply of fibrinogen, FV,
43
and other necessary coagulation factors. The resulting thrombin finally converts
fibrinogen to fibrin.
Procedure of PS assay:
The citrated plasma was obtained by the method described before. When removing the
citrated plasma, care should be taken to ensure that no platelets withdrawn. The PSactivity was determined by measuring the clotting time using a fully-automated Dade
Behring company, Marburg, Germany, BCs coagulation system was used. The
reference intervals were obtained as 65 -140%.
2.3.4 Screening test for lupus anticoagulants (LA1):
Principle of (LA1) method:
Russell's viper venom directly activates factor X. The test is not affected by contact
factor abnormalities or by factor VIII deficiencies or the presence of antibodies. A
circulating anticoagulant is usually indicated by a prolonged clotting time result that not
corrected by mixing patient plasma with normal plasma (substitution method).
Additional phospholipids are present in LA2 confirmation reagent to neutralize LA.
Procedure for LA assay:
Citrated plasma was obtained by the same coagulation standard method for blood
collection, preparation, and storage.
Manual procedure for LA1 assay:
Dispense 200 µl of the test plasma into a glass test tube (12x75) and warm for one
minute at 37 0C. Then add 200 ul of pre-warmed LA1 screening reagent or LA2
44
confirmation reagent to the plasma. Measure the clotting time. Repeat the test and
report the average as the result.
Automated assay method of LA1:
Special assay protocol for an automated clotting analyzer from Dade Behring Company,
Marburg, Germany, BCs coagulation system had been carried out. LA controls high and
low were determined. The normal range of LA assay was established as 31- 44 seconds.
If the clotting time of LA1 is within the normal range, no further testing is necessary. If
the LA1 clotting time is prolonged, further confirmatory test should be carried out.
2.3.5 Fibrinogen assay:
Principle:
Fibrinogen can be quantitatively measured by modification of thrombin time (TT)
test. The method involves clotting dilutions of both patient and control plasma with an
excess of thrombin. The coagulation time depends largely on the fibrinogen content of
the specimen.
Procedure of fibrinogen assay:
Preparation of the calibration curve:
Prepare the dilutions of fibrinogen standard with Owren’s buffer as follows: 1:5, 1:15,
and 1:40. Make all transfers from the first tube.
1:5 dilutions (first tube): 1.6 ml buffer to 0.4 ml fibrinogen standard.
1:15 dilution (second tube): 0.8 ml buffer to 0.4 ml from the first tube.
1:40 dilution (third tube): 2.8 ml buffer to 0.4 ml from the first tube.
Perform duplicate determinations on each dilution as follows:
45
Incubate 0.2 ml fibrinogen standard dilution at 37 0C for at least 2.0 minutes but not
more than 5.0 minutes. Add 0.1 ml thrombin reagent. Measure the clotting time twice.
Average the values. Plot the clotting time in seconds on the vertical axis versus the
concentration of fibrinogen standard dilutions on the horizontal axis.
Procedure for fibrinogen assay:
Prepare a 1:10 dilution of the test plasma and control using Owren’s buffer as follows:
0.1 ml plasma to 0.9 ml buffer. Incubate 0.2 ml of sample dilution at 37 0C for 2.0
minutes. Add 0.1 ml thrombin. Measure the clotting time and average the values.
Read the result from the calibration curve and record in mg/dL. The normal values
range from 200 to 400 mg/dL.
2.3.6 von Willebrand factor (vWF) assay:
Principle:
vWF (ristocetin-cofactor) from the sample causes agglutination of stabilized platelets
(provided by the von Willebrand reagent) in the presence of ristocetin. The resulting
agglutination decreases the turbidity of the reaction mixture. A coagulation instrument
measures the change in the absorbance and automatically determines the sample's
ristocetin cofactor activity in per centage.
Procedure for (vWF) assay:
Citrated plasma was used to determine the vWF using a fully automated coagulation
analyzer from Dade Behring Company, Marburg, Germany, BCs coagulation system.
The reference range established in the laboratory was 67-111%.
46
2.3.7 Activated Partial Thromboplastin Time (aPTT):
Principle:
The aPTT is a screening test used to assay all the plasma coagulation factors with the
exception of factors VII, XIII and platelets factor III (PF3). Optimal activation is
achieved by the addition of a platelet phospholipids substitute, which eliminates the
test's sensitivity to platelet number and function, as well as the addition of activators
such as kaolin, which eliminates the variability of activation by glass contact. The
aPPT is also used to monitor heparin therapy.
Procedure of APTT:
The citrated plasma was collected by the coagulation standard method for blood
collection, preparation, and storage.
The test was performed according to manufacturer's package insert. All tests on both
control and test plasma were performed in duplicates. A fully automated coagulation
analyzer from Dade Behring Company, Marburg, Germany, BCs coagulation system
was used.
2.3.8 Complete blood count (CBC):
The blood samples were collected in EDTA anticoagulant and tested for CBC
immediately using a fully automated haematology analyzer from Beckman/Coulter
Company, Miami, USA, STKS Model.
47
Chapter 111
3.0 Results:
The study involved two groups of women: pregnant women (n= 40) in third trimester,
ages between (22 and 38) years with medium age of 30 years and a control group of
non-pregnant women (n= 120) ages between (20 and 38) years with a medium age of
29 years.
3.1 Haematological tests:
The majority of pregnant women had low RBCs count (66%), low Haemoglobin
(91%) and low PCV (91%). Sixteen per cent had low platelets count, that was low as
94X103/µL in some pregnant women (Pregnancy-induced thrombocytopenia PIT).
The mean MCV is within normal value. Pregnant with microcytic RBCs= 18%.
(Tables 3.1, 3.2 & Fig.1). The RBCs count (p=0.001), Hb (p=0.001), PCV (p= 0.001),
PLT (p=0.007), were statistically significantly reduced during pregnancy, while MCV
(p= 0.2), MCHC (p= 0.3), MCH (p= 0.10), TWBCs (p=0.7) showed no significant
differences in the pregnant women compared to controls (Table 3.2; Fig. 1).
3.2 Haemostatic variables:
The differences in haemostatic variables between the two groups, pregnant women
and controls were illustrated in table 3.3.
LA1 and APTT times were within normal ranges with no significant difference
between the study groups, p=0.2 & 0.9 respectively. Fibrinogen levels and vWF were
significantly elevated in pregnant women compared to controls, p<0.001 & <0.01
respectively. PS was significantly reduced in pregnant women compared to controls
48
with p<0.001, while PC and ATIII were comparable in pregnant women and controls,
(p= 0.5 and p=0.9) respectively. Table 3.3 & Fig. 3.2.
No thrombotic incidence was recorded in pregnant women during the late pregnancy
or the puerperium.
In the puerperium, PS levels increased to reach normal levels in pregnant women and
were not statistically different compared to controls. While fibrinogen and vWF levels
dropped significantly to reach control levels. LA1 and APTT remained unchanged
(Tables 3.3, 3.4 & Fig. 3.3).
49
Table 3.1 Percentage of pregnant women with low and very low Haematological
parameters:
Haematological
RBCs
Hb
PCV
MCV
MCH
MCHC
PLT
WBCs
Low values
40%
73%
73%
29%
37%
16%
8%
5%
Very Low values
26%
18% 18%
23%
30%
52%
8%
3%
parameters
RBCs = red blood cell; Hb= haemoglobin; PCV= packed corpuscular volume;
MCV= mean corpuscular volume; MCH= mean corpuscular haemoglobin;
MCHC= mean corpuscular haemoglobin concentration; PLT= platelet; WBCs= white
blood cells;
Low values= ~SD below normal range; very low values = -2SD
50
Table 3.2 Haematological parameters for controls and pregnant women.
Haematological Parameters
Controls(n=120)
Mean ±S.D
(Range)
Pregnant women(n=40)
Mean ±S.D
(Range)
4.55±0.3(4.02-5.25)
4.14 ± 0.4 (3.51-5.11)
0.001*
Hb g/dl
13±0.97 (10.8-14.3)
11.2± 1.2 (8.7 - 13)
0.001*
PCV %
38±2.6 (32-41)
33.6±3.4 (26-39)
0.001*
MCV/ fl
83.9±5.7 (67-90)
81.3±6.9 (67-99)
0.2
MCH / pg
28±2.3 (22-31)
27±2.4 (22-31)
0.1
MCHC %
33.5±0.8 (32-35)
33.2±0.6 (29-35)
0.2
PLT (10 3 / cmm)
321±73.5(221-494)
256±70.8 (94-413)
0.007*
WBCs (10 3 / cmm)
7.9±2.5(3.6-13.2)
7.6±2.2 (3.7- 15.7)
0.7
RBCs(10 6/ cmm)
Level of significance: p< 0.05 was considered significant.
51
P- value
Haematologic results for controls
and pregnant women
40
35
30
25
Controls
Preg.
20
15
10
5
0
1
RBCs
2
Hb
3
4
PCV
WBCs
Fig. 3.1
52
Table 3.3 Haemostatic variables of controls and pregnant women.
Variables
Controls (n=120)
Mean ±S.D (range)
Pregnant women (n=40)
Mean±S.D (range)
p-value
PS level (%)
64.5±12.3 (33-94)
39.2±9.8 ( 20-56)
0.001*
PC level (%)
114±17.4 ( 83-143)
105±16 ( 66-145)
0.5
ATIII (%)
Level
106±11.8 (87-132)
108 ±13 (87-133)
0.9
440±91.2 (142-500)
0.001*
Fibrinogen (g/dl) 316±84.4 (145-500)
level
vWF (%)
Level
94.4 ±33 (55-150)
114 ±32.2 (46-149 )
0.01*
LA 1 (seconds)
35.9±4.75 (27-49)
37.2±4.76 (26-46)
0.2
APTT(seconds)
29.7±2.1 (26-34)
29.8±4.17 (21-40)
0.9
PS= Protein S; PC= Protein C; ATIII=Anti-thrombin III; vWF=vonWillibrand factor
LA1=lupus anti-coagulant; APPT= activated partial thromboplastin time;
53
Levels of Natural anticoagulants in
Controls & Pregnant women
120
100
%
80
Controls
Preg.
60
40
20
0
1
2
PS
PC
3
AT111
Fig. 3.2
54
Table 3.4 Levels of coagulation cascade proteins and coagulation tests during
pregnancy and puerperium.
Variables
During pregnancy
(n=40) Mean ±S.D
In the Puerperium (n=40)
Mean ±S.D
p-value
PS level (%)
39.2 ±9.8 (20-56)
66.1±11.9 (43-82)
0.001*
PC level (%)
105 ±16 (66-145)
117 ± 16.6 (83-143)
0.40
ATIII level (%)
108 ±13 (87-1 33 )
101 ±12.3 (73-125)
0.50
Fibrinogen level
(g/dl)
440 ±91.2 (142-500)
374 ±79.5 ( 246-501)
0.002*
vWF level (%)
114 ±32.2 (46-149 )
94.4 ±33 (55-150)
0.01*
LA 1 (seconds)
37.2 ± 4.8 (26-46)
36.9± 4.0 ( 28-43)
0.70
APTT(seconds)
29.8 ±4.2 (21-40 )
30.6 ±2.7 (25-34)
0.30
55
Natural anticoagulants levels in
controls, pregnant women and in
the puerperium
140
120
100
Controls
Pregnant
Puerperium
80
60
40
20
0
1
2
PS
PC
3
AT111
Fig. 3.3
56
Chapter 1V
4.0 Discussion & conclusion:
Pregnancy is a patho-physiological process that affects women with generalized
disturbance of all body systems; coagulation and fibrinolysis are no exceptions. The
haemostatic balance is tipped towards coagulation with marked and significant
increase in the fibrinogen and vWF. The increase in fibrinogen and vWF could be
explained by increased synthesis of these two coagulation acute-phase proteins. They
increase during stress, pregnancy or after surgery.101 This makes diagnosis or
evaluation difficult in patients suspected of having vWD. Therefore, it may be
necessary to study these patients on multiple occasions to rule out vWD. This may
probably point more towards the pathological effect of pregnancy in organs like the
liver and vascular endothelium. Normalization of the levels in the puerperium further
confirms this effect.
In pregnancy, the combination of low proteins S with increased fibrinogen and vWF
pose definite risk for thrombophilic incidents. Proteins C, S and ATIII may be
markedly reduced or at least show reduction to the lower range of normal.102 Maiello
M et al., (2006) 103and Galit, et al. (2005),104 reported similar results for significantly
decreased PS levels after the third trimester of normal pregnant women.
Fibrinogen, vWF and protein S levels normalized in the puerperium, indicating the
alleviation of the patho-physiologic effect of pregnancy. Some studies confirmed
activation of the fibrinolytic system as indicated by increased fibrinogen degradation
products (FDPs).
105
This fibrinolytic affect seemed to be localized in the uterus,
because it did not affect the fibrinogen level that continued to increase during
pregnancy. 106
57
The data for ATIII and PC was in keeping with those of other authors. Riordan MN,
107
(2003), Wickstrom et al., (2004)
108
reduced during the third trimester.
found that ATIII stays unchanged or slightly
Clark et al., (1998)109 and Cerneca et al.,
(1997)110have established that the PC remains unchanged or slightly increased
throughout pregnancy.
The low protein S, high fibrinogen and von Willibrand factor were in agreement with
the reports of Hellgren, M. (2003) 111 , and Borrelli, A., et al(2006) 112 in Caucasian
women. Jordaan DM, et al (2005) 113reported similar finding for black women with
reductions in PC and ATIII. No such study was conducted in expatriate pregnant
Sudanese women.
The normal levels of APTT, ATIII in the study pregnant women compared to the
controls most probably reflect the small number of our study groups. Larger sample
size may detect changes that are reported in pregnant women previously.
Haemoglobin and PCV were generally reduced starting from the second trimester,
probably due to increase plasma volume (haemo-dilution) as indicated by the normal
MCV; which may have an anti-thrombotic effect. This affect was clearly seen in our
study group as shown by a normal MCV in most of our pregnant women. On the other
hand some women were macrocytic, which has been attributed in some studies to
folate deficiency and sometimes to pregnancy alone. Some women in our group were
hypochromic and microcytic which is expected, although most of the women included
were from an affluent background.
Pregnancy induced thrombocytopenia (PIT) was seen in a small per centage of our
study group, no other factor could be thought of to explain the low normal/ low
platelets counts that was seen in less than a quarter of our study group.
58
The haemo-dilution effect of pregnancy was previous documented by DanilenkoDixon, D., et al (2001)114 Nikos K, et al (2001) 115
Pregnancy-induced thrombocytopenia (PIT) is a known phenomenon that was
previous described in a number of reports; Hisanori M and Ikuo S. (2000),116
Susanna S, et al (2000)117 Levy JA & Murphy LD (2002),118 Mamoru M, (2005)119
and WIN N, et al (2005).120
This study gives important information about changes in plasma levels of haemostatic
variables during normal pregnancy reflecting the status of coagulation.
None of the pregnant women in this study developed any thromboemboic disease or
pregnancy-related complications during pregnancy or puerperum.
The LA1, which is a known risk factor for thrombotic episodes,
121,122
was not
detected in our study women.
In conclusion, reduced proteins S, increased fibrinogen and vWF were clearly shown
to increase in the study pregnant women. Normal levels of protein C and ATIII could
simply reflect the small size of the study women. Although some study women
showed factors that predispose them to thrombosis, no thrombotic episodes were
reported in our study.
Recommendations:
Future large prospective studies are urgently needed to validate the use of current
approaches and perhaps define safer and more accurate strategies to reduce maternal
thrombotic episodes during pregnancy and after delivery.
Pregnant women with personal or family history of DVT, Placental abruption, Preeclampsia, Recurrent miscarriage and IUGR, should be considered for thrombophilia
screening and antenatal prophylaxis.
59
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