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CHAPTER 5
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done to rule out primary hepatic dysfunction which may affect the Protein C levels. The median values obtained in HbS patients for ALT and total Protein were higher than in the control subjects while the median value for Albumin was higher in the control. However none of the differences between these values reached statistical significance .The median INR value of the HbS patients compared with that of the control group was not statistically significant. In this study there is no evidence to support the fact that the lower Protein C level found in HbS compared to control is due to hepatic disease because there was no abnormality of ALT values in these patients with Sickle cell anaemia which is specific for hepatic injury.
There is evidence that patients with SCA have a state of persistent but low grade inflammation. Acute phase reactants such as C-reactive protein and serum amyloid A are moderately increased in sickle cell patients during steady state and significantly increased during painful vasoocclusive crisis. (Arkin et al, 1991) [51]. Elevated IL-1 and TNF - were found in patients with SCD during steady state. (Wick TM et al and Swerlick RA et al, 1993) [52].
Von Willebrand Factor a marker of injury to the endothelium and an acute phase reactant was found to be significantly increased in HbS patients in steady state compared with the HbA control and this value increased further during bone pain crisis.(J.B Schnog et al 2004)[14] and Iwara and Shokunbi, 2009.[53]
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Elevated acute phase reactants during the steady state suggest that patients with SCD have subclinical microvascular ischaemic events that lead to tissue injury, release of inflammatory cytokines and hepatic synthesis of acute phase reactants.
Nsiri et al, 1996 [44] and Fakunle,Shokunbi and Iwara , 2009[54] found significant increase in D-D dimer levels in HbS patients in steady state compared with Hb A healthy individuals. This confirms the activation of coagulation and fibrinolytic systems in steady state .The consensus regarding activation of coagulation in patients with Sickle cell disease are that there is in vivo generation of thrombin even in steady state. Nash et al, 1984 [18] and Clark MFR et al, 1980[19].
The reduced level of Protein C found in HbS patients in this study and other previous studies may be a consequence of chronic consumption of Protein C due to increased thrombin generation and activation of coagulation factors.
The study by Karayalin and Lanzkowsky in 1989 [13]found that the low Protein C levels in HbS patients in steady state decreased further during vasooclusive crisis. This underscores the increased consumption of Protein C during VOC and increased risk of thomboembolism in these patients.
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The underlying pathophysiology of thrombophilia in Sickle cell disease is a multifactorial process. Further studies should focus on comparing Protein C in steady state and in crisis in Nigerian sickle cell disease patients.
CONCLUSION
From this study Protein C activity is found to be lower in HbS patients in steady state compared with HbA Control population.
The low Protein C level observed in the HbS patients may not be due to hepatic dysfunction as Liver function tests in the subjects were found to be normal.
RECOMMENDATIONS
1. The use of warfarin in HbS patient for prophylaxis should be with caution as this will further lower the Protein C level, being a Vitamin K dependent
inhibitor of coagulation.
2. The HbS patient scheduled for major surgery should be given heparin in prophylactic doses so as to alleviate the risk of thromboembolic disorder.
3. The HbS patients with severe VOC should have heparin in prophylactic doses in view of the hypercoagulable state, as this may shorten the length of the vaso-occlusive event.
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4. The use of Protein C in the treatment of sickle cell disease should be
advocated especially when the patients require oral anticoagulation as protein C has been found to have anti inflammatory and fibrinolytic effects.
Complications of bleeding tendency, toxicity and other general side effects of currently available anticoagulant agents can also be avoided.
5. Serial annual monitoring of Protein C in HbS patients.
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REFERENCES.
1. Stuart J, Nash G.B. Red cell deformability and haematological disorders.
Blood review 1990; 4: 141-7.
2. Herrick J B. Peculiar elongated and Sickle shaped red blood corpuscles in a case of severe anaemia. Arch. Intern. Med. 1910; 6:517- 521.
3. Drayna D. Founder mutations. Scientific Americans 2005; 953: 60-7.
4. Weatherall DJ & Clegy J B. Inherited disorders: an increasing global health problem. Bulletin WHO 2001; 79: 704-12.
5. Nagel RL. Malaria and hemoglobinpathies. In: Steinberg MH. Disorders of haemoglobin. New York: Cambridge University Press; 2001. 832-60.
6. Pauling L, Itano H, Singer SJ, & Wells IC. Sickle cell anaemia: a molecular disease Science 1949; 110: 543-65.
77
7. Briehl, R.W & Nikolopoulou P. Kinetics of hemoglobin S polymerization and gelation under sheer: I. shape of the viscosity progress curve and dependence of delay time and reaction rate on shear rate and temperature blood 1993; 81: 2420-8.
8. Dobbe Jaa, Hardeman MR, Strekstra GJ, Strackee J, Ince C, &
Grimbergen CA, Analysing red blood cell-deformability distributions, Blood Cells, molec and Dis 2002; 28: 373-84.
9. Neuro-ophthalmological sequelae of sickle cell disease. West Afr.J Med.1990 Oct- Dec; 9(4):317-20.
10. Ballas Sk, Mohandas N. Sickle red cell microrheology and sickle blood rheology. Microcirculation 2004; 11: 209-25.
11. Pediatric International Dec 2001; 43 (6): 592-6.
12. Marfaing-Koka, et al; nouv Rev Fr Hematol 1993; 35: 425-430.
13. Karayalcin, et al; The American Journal of Pediatric Haematology/oncology 1989; 11(3): 320-323.
14. J.B Schnog et al; Protein C and S and inflammation in Sickle cell disease.American Journal of Haematology 2004; 76:26-32.
15. American Society of Haematology Self Assessment Programme Textbook.3rd Ed; 2007; 365-366.
78
16. Akinyanju OO. A profile of sickle cell disease in Nigeria. Ann NY Acad Sci; 1989; 565: 126-136.
17. Wheters DL, Ramirez Gm, Koshy m, Steinberg MH & Philips G et al, Accelerated healing of chronic sickle-cell leg ulcers treated with RGD peptide matix. RGD study group. The American Society of Haematology; 1994; 84: 1775-1779.
18. Nash GB, Johnson CS & Meiselman Hj. Mechanical properties of oxygenated red blood cells in sickle cell (HbSS) disease. Blood; 1984;
63: 73-80.
19. Clark MFR, Mohandas N & Shohet Sb. Deformability of oxygenated irreversible sickled cells. J Clin Invest; 1980; 65: 189-196.
20. Schmalzer EA, Manning RS, Chien S. Filtration of sickle cells:
recruitment into a rigid fraction as a function of density and oxygen tension. J Lab Clin Med.; 1989; 113: 727-734.
21. Makie LH & Hochmuth RM. The influence of oxygen tension, temperature and hemoglobin concentration on the rheological properties of sickle erythrocytes. Blood; 1990; 76: 1256-1044.
22. Kaul DK, Xue H. Rate of deoxygenation and rheologic behaviour of blood in sickle cell anemia. Blood; 1991; 77: 1353-1361.
79
23. Busari FI, January. Coagulation profile in patients with SCA, M.Sc Haematology dissertion, University of Ibadan. 1991.
24. Falusi AG, Esan CJF. Foetal haemoglobin levels in sickle cell aneamia in Nigeria. Afr. J. Med. Sci. 1989; 18: 145-149.
25. Famodu AA, Halim NKD, Odoemene DI, Eneh EE. Haematological changes in sickle cell disease: Measurement and significance; British Journal of Biomedical Science. Dec 1998.
26. Ohene-Frempong K, Nkrumah FK. Sickle cell disease.Africa Raven Press Ltd., New York: In: Embury Sh, Hebbel Rp, Mohandas N, Steinerg MH, eds. 1994.
27. Hickman M, Modell B, Greengross P. Mapping the prevalence of sickle cell and beta thalassaenia in England: estimating and validating ethnic- specific rates. Br J Haematol; 1999. 104: 860-867.
28. Lard LR, Mul FPJ, Hass M, Roos D, Duits AJ. Neutrophil activation in sickle disease. J Leucocyte Biol.; 1999; 66: 411-414.
29. Liesner R, Mackie I, Cookson J. Prothrombotic changes in children with sickle cell disease: relationships to cerebrovascular disease and transfusion. Br J Haematol; 1998; 103: 1037-1044.
80
30. Zwaal RFA, Comfurius P, Bevers EM. Mechanism and function of changes in membrane phospholipids asymmetry in platelets and erythrocytes. Biochem Soc Trans. 1993; 21: 248-253
31. Frank PFH, Bevers EM, Lubin BH. Uncoupling of the membrane skeleton from the lipid bilayer: the cause of accelerated phospholipid flip-flop leading to an enhanced procoagulant activity of sickle cells. J.
Clin Invest. 1985; 75: 183-190.
32. Rosing J. Van Rijn JL, Bevers EM, Van Dieijen G, Comfurius P et al, The role of activated human platelets in prothrombin and factor X activation. Blood. 1985; 65: 319-332.
33. Manodori AB, BA rabino GA, Lubin BH, Kuypers FA. Adherence of phosphatidylserine-exposing erythrocytes to endothelial matrix thrombospondin. Blood; 1999; 95: 1293-1300.
34. Bayazti AK, Klinic Y. Natural coagulation inhibitors Protein C, protein S, antithrombin in patients with sickle cell anemia in a steady state;
Pediatr Int. Dec 2001; 43(6): 592-596.
35. Devine DV, Kinney TR, Thomas PF,. Fragment D-Dimer levels: an objective marker of vaso-occlusive crisis and other complications of sickle cell disease. Blood; 1986; 68: 317-9.
81
36. Solovey A, Gui L, Key NS, Hebbel RP. Tissue factor expression by endothelial cells in sickle cell disease. J Clin Invest.; 1998;101: 1899-1904
37. Westerman MP, Green D, Gilman-Sachs A. Antiphospholipid antibodies, protein C and S, and coagulation changes in sickle cell disease. J Lab Clin Med.; 1999; 134: 352-362.
38. Francis RB. Protein S deficiency in sickle cell anaemia. J. Lab clin Med;
1988; 111:571-576.
39. Edington TS, Mackman N, Brand K, Ruf W. The structural biology of expression and function of tissue factor. Throm Haemost.; 1991; 66: 67-79.
40. Kaul DK, Hebbel RP, Hypoxia/ reoxygenation causes inflammatory response in transgenic sickle mice but not in normal mice. J Clin Inves.
2000; 106: 411-420.
41. Stuart MJ, Setty BNY. Hemostatic alteration in sickle cell disease:
relationships to disease pathophysiology. Pediatr Pathol Mol Med.; 2001;
20: 27-46.
42. Wun T, Paglieroni T, Tabin F, Welborn J, Nelson K et al. Platelet activation and platelet-erythrocyte aggregates in patients with sickle cell disease. J Lab Clin Med.; 1997; 129: 507-516.
82
43. Key NS, Shingaard A, Dandlet L. Whole blood tissue procoagulant activity is elevated in patients with sickle cell disease. Blood. 1998; 91 4216-4223.
44. Westerman MP, Green D, Gilman-Sactis A, et al. Antiphospholipid antibodies, protein C and S and coagulation changes in sickle cell disease. J Lab Clin Med. 1999; 134: 352-362
45. Wright JG, Matia R, Cooper P, Thomas P, Preston FE, Serjeant GR.
Protein C and S in homozygous sickle cell disease: does hepatic dysfunction contribute to low levels? Br J Hematol. 1997; 98: 627-631.
46. Lane PA, O’Conell JL, Marler RA. Erythrocyte membrane vesicles and irreversible sickle cells bind protein S. Am J Hematol 1994; 47: 295-300.
47. Tam DA. Protein C and S activity in sickle cell disease and stroke. J child Neurol. 1997; 12: 19-21
48. Leichtman DA, Brewer GJ, Elevated plasma levels of fibrinopeptide A during sickle cell anemia pain crisis-evidence for intravascular coagulation. AM J Hematol 1978; 5: 183-190
49. Mahmod A, Macintosh DM, Sharper AG. Fibrinolytic activity in the clinical crisis of sickle-cell anemia. Br Med J 1967; 3: 5 653-4.
50. Gordon PA, Breeze Gr, Mann JR, Stuart J. Coagulation fibrinolysis in sickle cell disease. J Clin Pathol 1974; 27:485-9.
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51. Arkin S,Naprtek B,Guarini L,1991.Expression of intracellular adhesion molecule(CD54) on hematopoietic progenitors. Blood; 77:948-53.
52. Wick TM, Moake JL, Udden MM, McIntire LV, 1993.Unusually large von Willebrand factor multimers preferentially promote young sickle and non sickle erythrocyte adhesion to endothelial cell. Am J Hematol; 4:84-92.
53. Iwara KU, Fakunle EE, Shokunbi WA.von Willebrand factor antigen as a prognostic index in sickle cell disease.The Nigerian Society for Haematology and Blood Transfusion conference abtract booklet 2009;pg8
54. Fakunle EE, Iwara K, Shokunbi WA. D Dimer levels in sickle cell disease patients during bone pain crises and in steady state. The Nigerian Society for Haematology and Blood Transfusion. Abstract booklet 2009.pg 4.
55. Mohsen AF, El-Hazmi, Warsy AS, Bahakin H. Blood proteins C and S in sickle cell disease. Acta haematologica.1993; 90(3): 114-9.
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APPENDICES CONSENT FORM
Title of the research:
Evaluation of protein C in Nigerian patients with sickle cell anaemia in steady state.
Name of applicant:
This study is being conducted by Dr. Ajayi Adeleke Ibijola.
Department of haematology U.C.H. Ibadan.
Sponsor of research:
This study is sponsored by Dr. Ajayi Adeleke Ibijola.
Department of Haematology, University College Hospital Ibadan.
Purpose of research:
To assess prothrombotic state in Nigerian patients with sickle cell anaemia in steady state.
Procedure of the research:
Each participant will give 9.5 ml of blood in all, for PT, APTT and protein C assay and LFT. We are planning to recruit 100 participants into this study from Haematology Day Care Clinic and medical Out –Patient Clinic of the U.C.H Ibadan.
Expected duration of research and of participants’ involvement:
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We expect participants to be involved in this research for 3 months.
Risks:
The 9.5 ml of blood is not harmful to an adult HbS in steady state.
Costs to the participants:
Your participation in this research will not cost you anything.
Benefits:
The research will assess the prothrombotic tendency in the patients and help to manage them better during vasooclusive crisis.
Confidentiality:
All information given will be treated with confidentiality.
Voluntariness:
Your participation in this research is entirely voluntary.
Alternatives to participation:
If you choose not to participate, this will not affect your treatment in this hospital in any way.
Due inducement:
You will be adequately informed of any abnormality detected and measures will be taken to ensure that appropriate management is instituted. However; you will not be paid any fees for participating in this research.
Consequences of participants decision to withdraw from research and Procedure for orderly termination of participation:
You can also choose to withdraw from the research at anytime.
Modality of providing treatments and actions to be taken in case of injury or adverse events:
If you suffer any injury as a result of your participation in this research, you will be treated at the University of Ibadan Teaching Hospital and the researcher will bear the cost of this treatment.
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STATEMENT OF PERSON OBTAINING INFORMED CONSENT:
I have fully explained this research to _______________________________
and have given sufficient information about risks and benefits to make an informed decision.
Date: _________________
Signature: ____________________________________
Name; ______________________________________________
STATEMENT OF PERSON GIVING CONSENT:
I have read the description of the research or have had it translated into language I understand .I have also talked it over with the doctor to my
satisfaction. I understand that my participation is voluntary. I know enough
87
about the purpose ,methods ,risks and benefits of the research study to judge that I want to take part in it .I understand that I may freely stop being part of the study at any time. I have received a copy of this consent form and additional information sheet to keep for myself.
Date: ________________________________
Signature: _______________________________
Name: __________________________________
Witness’ Signature (if applicable) _________________________________
Witness Name (if applicable) ________________________________
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QUESTIONNAIRE
Questionnaire on the study of evaluation of Protein C in Nigerian patients with sickle cell anaemia in steady state. (To be administered by the principal investigator)
This questionnaire is part of a study to evaluate protein C in Nigerian patients with sickle cell anemia in steady state so as to assess their pro-thrombotic tendency compared with normal HbA individuals and its contribution to morbidity and mortality.
SECTION A Bio-data section (Fill in as appropriate or tick)
1. Serial Number: _____________________________________
2. Name: ____________________________________________
3. Age: _____________________________________________
4. Sex: _____________________________________________
5. Address ____________________________________________
6. Phone No. __________________________________________
7. Occupation __________________________________________
(i) Student (indicate level) ______________________
(ii) Professional (Specify type) _______________
(iii) Job applicant
(iv) Artisan
(v) Petty Trader 8. Educational Achievement
(i) No formal Education
(ii) Up to primary school
89 (iii) Up to secondary school (iv) University Education
9. Religion
(i) Christianity
(ii) Muslim
(iii) Paganism
(iv) Others (specify) ______________________________
SECTION B
1 Haemoglobin Electrophoresis
2. Age at diagnosis of sickle cell disease 3. Last episode of crisis
4. No of bone pain in a year
5. Have you been transfused before? (a) Yes (b) No
6. No of transfusion till date (a) 0 (b) 1 (c) 2 (d) 3 (e) ≥ 4
7. How many admissions since the past one year (a) 0 (b) 1 (c) 2 (d) 3 (e) ≥4
8. Have you ever had a leg ulcer which did not heal in 2 months?
(a) Yes (b) No 9. If yes in NO. 8 how long did it take? (a) >2-5 months (b) >5-9 months (c) 21-25%
(d) 26-30% (e) ≥ 31%
10. What is your steady state PCV? (a) 10-15% (b) 16-20% (c) 21-25% (d) 26-30%
(e) ≥ 31%