Saudi Journal of Gastroenterology
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ORIGINAL ARTICLE Table of Contents   
Year : 2007  |  Volume : 13  |  Issue : 2  |  Page : 76-80
Hepatitis C virus-associated thrombocytopenia is not related to serum thrombopoietin levels


1 Department of Clinical Pathology, Faculty of Medicine, Assuit University, Assiut, Egypt
2 Department of Internal Medicine, Faculty of Medicine, Assuit University, Assiut, Egypt
3 Department of Tropical Medicine and Gastroenterology, Faculty of Medicine, Assuit University, Assiut, Egypt

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   Abstract 

Objective: Hepatitis C virus (HCV) infection is one of the most important health problems in Egypt. Thrombocytopenia is a common finding in subjects with chronic hepatitis. The precise etiology of this thrombocytopenia is still obscure. There is increasing interest in the potential role of thrombopoietin (TPO) as a cause of this thrombocytopenia. The aim of this work was to determine serum TPO levels in HCV-positive patients and to test the assumption that HCV-associated thrombocytopenia could be due to low TPO levels. Materials and Methods: Forty patients with HCV infection were included in this study and classified into three groups according to the degree of thrombocytopenia (IA-mild, IB-moderate, II-none). Twenty five healthy volunteers served as control (Group III). All patients and controls had undergone full clinical assessment and the following laboratory investigations: complete blood count, liver function tests, prothrombin time and concentration and serum TPO level. Results: TPO levels were significantly elevated in Group IA as compared to the control group ( P <0.05). No significant difference was found between groups IA and II. TPO in Group IB was slightly, but insignificantly reduced compared to Group IA but did not differ statistically from the control or Group II. Significant negative correlation was found between serum TPO levels and platelet counts in Groups IA, IB and II (r=-0.421, P <0.05). No correlations were found between serum TPO levels and liver function tests or hematological parameters. Conclusion: An impaired TPO production did not explain the development of thrombocytopenia in HCV and other mechanisms must be involved.

Keywords: Hepatitis C, thrombocytopenia, thrombopoietin.

How to cite this article:
Afifi OA, Sewify EM, El-Attar MM, Taie AO, Mostafa AK. Hepatitis C virus-associated thrombocytopenia is not related to serum thrombopoietin levels. Saudi J Gastroenterol 2007;13:76-80

How to cite this URL:
Afifi OA, Sewify EM, El-Attar MM, Taie AO, Mostafa AK. Hepatitis C virus-associated thrombocytopenia is not related to serum thrombopoietin levels. Saudi J Gastroenterol [serial online] 2007 [cited 2020 Oct 29];13:76-80. Available from: https://www.saudijgastro.com/text.asp?2007/13/2/76/32181



   Introduction Top


The population of Egypt has a heavy burden of liver disease, mostly due to chronic infection with hepatitis C virus (HCV). The overall prevalence of antibody to HCV in the general population is around 15-20%. The risk factor for HCV transmission that specifically sets Egypt apart from other countries is the high rate of exposure to HCV as a consequence of parenteral anti-schistosomal therapy. [1] Thrombocytopenia is a common finding in subjects with chronic hepatitis. However, controversy still exists concerning the mechanism of HCV-associated thrombocytopenia. It can occur either due to a decrease in platelet production by megakaryocytes, an increase in peripheral destruction or increased splenic sequestration. [2]

The significantly lower platelet counts in patients with cirrhosis of viral origin indicate that some factors other than thrombopoietin (TPO) may contribute to the thrombocytopenia, e.g., a direct viral effect on megakaryocytes and anti-platelet antibodies. [3] TPO, the main stimulus for thrombopoiesis regulates platelet production stimulating megakaryocyte proliferation and maturation. It is produced primarily in the liver and degraded by circulating platelets. [4] The aim of this work is to determine serum TPO levels in an attempt to evaluate thrombopoiesis in HCV-positive patients with and without thrombocytopenia and to test the assumption that HCV-associated thrombocytopenia could be due to low TPO levels.


   Materials and Methods Top


Forty patients with HCV infection were enrolled in this study. They were diagnosed as having chronic HCV at an early stage of the disease based on: clinical data, ultrasonographic findings, liver function tests, polymerase chain reaction (PCR) for HCV and histopathological study of liver tissue obtained by biopsy.

The PCR test was done as follows: nucleic acid was extracted using an automated extraction system. HCV detection and quantitation were performed using the Abbott Real-Time M2000 rt instrument. The assay utilizes two distinct sets of primers and probes. The first set targets a conserved region of the 5' untranslated region of the HCV genome, while the second set is specific for an internal control (IC) that is processed with each sample to control for sample recovery and inhibition. The lower limit of detection of the PCR test used is 3200 copies/mL.

Exclusion criteria were HCV patients with splenomegaly (clinically or ultrasonographically), hypersplenism, liver cirrhosis, relevant diseases of other organs or systems such as heart failure, coronary artery disease, renal insufficiency and lung disease or malignancy.

Patients were 29 males and 11 females. Their ages ranged from 24 to 62 years with a mean ± S.D. of 43.16 ± 9.1 years. None of these patients was on or was previously treated with interferon or any other anti-viral medication. Patients were classified into:

Group I: HCV patients with thrombocytopenia (25 patients) and they were subdivided into:

Group IA: (8 patients) with mild thrombocytopenia (platelet count > 100 x 109/L).

Group IB: (17 patients) with moderate thrombocytopenia (platelet count < 100 x 109/L).

Group II: HCV patients without thrombocytopenia (15 patients).

In addition, 25 apparently healthy age- and sex-matched volunteers were included as controls (19 males and 6 females). Their ages ranged from 18 to 62 years with a mean ± S.D. of 42.8 ± 9.5 years. All healthy control subjects were HCV antibody-negative.

All patients and controls had undergone full clinical assessment and the following laboratory investigations: complete blood count (CBC) performed with cell-Dyn 3500 (Abbott diagnostics, USA), liver function tests done using an automated chemistry analyzer (BM/Hitachi-911), prothrombin time (PT) and concentration (PC) performed by ST art coagulometer using Neoplastin Cl plus supplied by Diagnostica Stago, France. Serum TPO levels were determined in serum stored at -20°C by sandwich enzyme-linked immunoSorbent assay (ELISA) using a commercially available ELISA kit (Quantikine Human TPO immunoassay) from RandD systems, USA. [5]

Statistical analysis

The results were reported as mean ± standard deviation (S.D.) and range as appropriate. Statistical comparisons were done with Student's t-test and one-way analysis of variance (ANOVA) for independent samples when comparing two and three groups respectively. Statistical significance was accepted for P<0.05 (significant), P<0.001 (highly significant). Relationships between variables were estimated by the Pearson's correlation coefficient (r).


   Results Top


[Table - 1] shows the CBC parameters in different groups. There was a highly significant reduction in platelet count in Group I as compared to both Group II and the Control group (P=0.001 for both). Similarly, plateletcrit (PCT) was significantly less in Group 1 compared with both Group II and the Control group (P=0.001 and P=0.02 respectively; [Table - 1]). Also, PCT was significantly higher in Group II compared to the Control group (P=0.001; [Table - 1]).

[Table - 2] shows the liver function tests in different groups. There was significant impairment in both Groups I and II compared to the control group (P<0.01). The impairment of total bilirubin, total protein and serum albumin was more pronounced in Group I than that in Group II (P<0.01, P<0.05 and P<0.05 respectively). Significantly, prolonged PT and reduced PC were found in both Groups I and II when each were compared with the control group (P<0.0001 for both; [Table - 2]). Also, there was significant prolongation of PT and reduction of PC in Group I when compared with Group II (P<0.05 and P<0.01 respectively; [Table - 2]).

The mean ± S.D. of TPO levels in Group I was 123.56 ± 24.92 pg/ml which did not differ statistically from other groups. [Table - 3] shows serum TPO levels in relation to the degree of thrombocytopenia in patients. TPO levels were significantly elevated in Group IA (patients with mild thrombocytopenia) when compared with the Control group (P=0.044). There was mild reduction of TPO in Group IB (patients with moderate thrombocytopenia) when compared with either Group IA or Group II (patients without thrombocytopenia), but with no significant difference (P>0.05). TPO level in Group IB didn't differ statistically from the control group (P>0.05).

In this study, a statistically significant negative correlation was found between serum TPO levels and platelet count in group I (P=0.036, r=-0.421; [Figure - 1]). No correlations were found between serum TPO levels and liver function tests or hematological parameters.


   Discussion Top


Infection with HCV is a major cause of chronic liver diseases. HCV antibody-positive individuals are 2.6 times more likely to have a low platelet count than those who are HCV-antibody negative. [6] Treatment of patients with HCV-associated thrombocytopenia is an important and unresolved problem particularly because the mechanism responsible for thrombocytopenia in these individuals remains unclear. [7] As TPO is mainly produced in the liver, it was postulated that inadequate TPO production might be responsible for the thrombocytopenia accompanying liver disease. [8]

In this study, CBC revealed highly significant reduction in platelet counts as well as PCT in group I individuals compared to both controls and group II. No statistical differences were detected concerning other CBC parameters. These findings are consistent with those of previous studies reported by Nagamine et al . [9]

In the current study, serum TPO levels in groups I and II showed no statistically significant difference when compared with the control group. Although the mean serum level of TPO in Group IB (HCV patients with moderate thrombocytopenia) was reduced to a greater extent than that in Group IA (HCV patients with mild thrombocytopenia), the difference was not of statistical significance. These results are consistent with those previously reported by Frank et al. [1] and Koruk et al. , [10] who found that thrombocytopenic patients with chronic hepatitis C viral infection presented with serum TPO levels similar to those of the healthy control group. In addition, Okubo and Kawasaki [11] reported that TPO levels were normal in non-cirrhotic HCV patients, suggesting its diminished production is not the cause of thrombocytopenia seen in these patients. Other studies also concluded that TPO did not prevent the development of thrombocytopenia. [12],[13]

On the other hand, in patients with HCV and mild thrombocytopenia (Group IA), the mean serum level of TPO was significantly elevated when compared with the control group. This finding was also supported by a previous study which reported that serum TPO levels were elevated in patients with an early stage of chronic viral hepatitis. As the disease progresses from mild fibrosis to cirrhosis, decreased production of TPO may contribute to the further development of thrombocytopenia in cirrhosis. [14]

In an attempt to explain the above findings, it was suggested that the production of TPO is maintained at certain levels until profound impairment of hepatic function has developed. However, the production of TPO by other organs compensates for the decrease in TPO production by the liver. [8]

In this study, no correlation was found between total bilirubin and TPO. These findings are in agreement with previous reports that demonstrated significant elevation in bilirubin in cases of HCV infection with thrombocytopenia but they could not find any statistical correlation between serum TPO levels and bilirubin. [8],[15] Also, there was no correlation between TPO and serum albumin and these results are consistent with those from the studies mentioned above. [8],[15]

In our study, we also found that there was no correlation between TPO and alkaline phosphatase or aminotransferases. This result is again consistent with that previously reported by Espanol et al. , [15] who have shown no statistical correlation between TPO and liver enzymes in HCV patients. However, Giannini et al. , [16] found that in patients with chronic HCV infection, serum TPO levels were correlated to liver functional impairment and to the degree of liver fibrosis.

No statistically significant correlation was found in this study between TPO serum levels and PT or PC. It was also revealed that PT is more prolonged in the presence of thrombocytopenia in hepatitis C virus infection with no correlation with TPO serum level. This finding was also reported by Espanol et al. [15] and Giannini et al . [17] In contrast, Kawasaki et al. , [14] identified in their study a significant positive correlation between TPO and PC in HCV patients.

In this study, we found a statistically significant negative correlation between serum TPO levels and platelet counts in group I (r= -0.396, P=0.042). This finding is consistent with that previously reported by Nagamini et al. , Wendling et al. , Hou et al. and Schoffski et al. , [9],[18],[19],[20] who found in their studies significant negative correlations between TPO serum levels and platelet counts. In contrast, Kawasaki et al. and Panasiuk et al. [2],[14] found positive correlations of TPO with platelet counts in HCV patients with thrombocytopenia and liver cirrhosis.

On the other hand, Espanol et al. and Aref et al. , [13],[15] reported that there was no correlation between TPO and platelet counts and concluded that serum TPO levels had no role in the occurrence of thrombocytopenia in chronic liver disease. Rios et al. , [4] also reported that serum TPO levels are not related to platelet counts in liver disease suggesting that impaired production of TPO occurs only in the failing liver and increased TPO degradation by platelets sequestered in the congested spleen may contribute to the thrombocytopenia in cirrhotic patients. In addition, other mechanisms involved in the development of thrombocytopenia with HCV infection, such as autoimmune mechanism and direct viral effect on megakaryocytes have also been reported. [4]

Potential limitations of the present study are the small number of patients included in each patient group which may cause a type 2 error when analyzing data and a lack of positive control group (subjects with reduced TPO levels). However, this study provides preliminary evidence that TPO levels alone may not contribute to thrombocytopenia in this patient group and highlights the need for a larger study. Also, the possibility of a role for anti-TPO antibodies or antagonists needs to be tested in in vivo TPO infusion-response studies.


   Conclusion Top


We conclude that serum TPO levels in patients with hepatitis C viral infection and thrombocytopenia did not differ significantly from those of healthy subjects, excluding a defect in TPO production in the early stages of the disease. Other mechanisms are possibly involved in the development of thrombocytopenia in these patients, such as peripheral immune-mediated platelets destruction, presence of anti-TPO antagonists or antibodies and/or a direct viral cytopathic effect on megakaryocytes.


   Acknowledgments Top


The authors would like to thank Dr. Ahmed Helmy, Lecturer of Tropical Medicine and Gastroenterology, Assiut University Hospital, Assiut, Egypt, for his efforts in writing, editing and publishing this article.

 
   References Top

1.Frank C, Mohamed MK, Strickland GT, Lavanchy D, Arthur RR, Magder LS, et al . The role of parenteral antischistosomal therapy in the spread of hepatitis C virus in Egypt. Lancet 2000;355:887-91.  Back to cited text no. 1  [PUBMED]  [FULLTEXT]
2.Panasiuk A, Prokopowicz D, Zak J, Panasiuk B. Reticulated platelets as a marker of megakaryocytopoiesis in liver cirrhosis: Relation to TPO and hepatocyte growth factor serum concentration. Hepatogastroenterology 2004;51:1124-8.  Back to cited text no. 2  [PUBMED]  
3.Vyzantiadis T, Theodoridou S, Giouleme O, Evgenidis N, Vyzantiadis A, Garipidou V. Serum thrombopoietin levels in thrombocytopenic patients with liver cirrhosis. Haematologica 2002;87:890-1.   Back to cited text no. 3  [PUBMED]  [FULLTEXT]
4.Rios R, Sangro B, Herrero I, Quiroga J, Prieto J. The role of TPO in the thrombocytopenia of patients with liver cirrhosis. Am J Gastroenterol 2005;100:1311-6.   Back to cited text no. 4  [PUBMED]  [FULLTEXT]
5.Tahara T, Usuki K, Sato H, Ohashi H, Morita H, Tsumura H, et al . A sensitive sandwich ELISA for measuring thrombopoietin in human serum: Serum thrombopoietin levels in healthy volunteers and in patients with hemopoietic disorders. Br J Haematol 1996;93:783-8.  Back to cited text no. 5  [PUBMED]  
6.Streiff MB, Mehta S, Thomas DL. Peripheral blood count abnormalities among patients with hepatitis C in USA. Hepatology 2002;35:947-52.  Back to cited text no. 6  [PUBMED]  [FULLTEXT]
7.Garcia-Suarez J, Burgaleta C, Hernanz N, Albarran F, Tobaruela P, Alvarez-Mon M. HCV-associated thrombocytopenia: Clinical characteristics and platelet response after recombinant a2b- interferon therapy. Br J Hematol 2000;110:98-103.  Back to cited text no. 7  [PUBMED]  [FULLTEXT]
8.Shiota G, Okubo M, Kawasaki H, Tahara T. Interferon increases serum trombopoietin in patients with chronic hepatitis C. Br J Haematol 1997;97:340-2.  Back to cited text no. 8  [PUBMED]  
9.Nagamine T, Ohtuka T, Takehara K, Arai T, Takagi H, Mori M. Thrombocytopenia associated with HCV infection. J Hepatol 1996;24:135-40.  Back to cited text no. 9  [PUBMED]  [FULLTEXT]
10.Koruk M, Onuk MD, Akcay F, Savas MC. Serum TPO levels in patients with chronic hepatitis and liver cirrhosis and its relationship with circulating thrombocyte counts. Hepatogastroenterology 2002;49:1645-8.  Back to cited text no. 10  [PUBMED]  
11.Okubo M, Shiota G, Kawasaki H. Thrombopoietin levels in serum and liver tissue in patients with chronic viral hepatitis and hepatocellular carcinoma. Clin Sci (Lond) 2000;99:207-14.   Back to cited text no. 11  [PUBMED]  [FULLTEXT]
12.Schiodt FV, Balko J, Schilsky M, Harrison ME, Thornton A, Lee WM, et al . Thrombopoietin in acute liver failure. Hepatology 2003;37:558-61.  Back to cited text no. 12    
13.Aref S, Mabed M, Selim T, Goda T, Khafagy N. Thrombopoietin (TPO) levels in hepatic patients with Thrombocytopenia. Hematology 2004;9:351-6.   Back to cited text no. 13  [PUBMED]  [FULLTEXT]
14.Kawasaki T, Takeshita A, Souda K, Kobayashi M, Suzuki F, Kageyama F, et al . Serum TPO levels in patients with chronic hepatitis and liver cirrhosis. Am J Gastroenterol 1999;94:1918-22.   Back to cited text no. 14    
15.Espanol I, Gallego A, Rabella N, Lerma E, Hernandez A, Pujol-Moix N. Thrombocytopenia associated with liver cirrhosis and hepatitis C viral infection: Role of thrombopoietin. Hepatogastroenterology 2000;47:1404-6.   Back to cited text no. 15  [PUBMED]  
16.Giannini E, Borro P, Botta F, Fumagalli A, Malfatti F, Podesta E, et al . Serum TPO levels are linked to liver function in untreated patients with HCV-related chronic hepatitis. J Hepatol 2002;37:572-7.  Back to cited text no. 16  [PUBMED]  [FULLTEXT]
17.Giannini E, Botta F, Borro P, Malfatti F, Fumagalli A, Testa E, et al . Relationship between TPO serum levels and liver function in patients with chronic disease related to HCV infection. Am J Gastroenterol 2003;98:2516-20.   Back to cited text no. 17  [PUBMED]  [FULLTEXT]
18.Wendling F, Cohen-Solal K, Villeval JK, Debili N, Andvainchenker W. MPL-ligand or TPO: Biological activities. Biotherapy 1998;10:269-77.  Back to cited text no. 18    
19.Hou M, Andersson PO, Stockelberg D, Mellquist UH, Ridell B, Wadenvik H. Plasma Thrombopoietin levels in thrombocytopenic status: Implicationfor a regulatory role of bone marrow megakaryocytes. Br J Haematol 1998;101:420-4.  Back to cited text no. 19    
20.Schoffski P, Tacke F, Trautwein C, Martin MU, Caselitz M, Hecker H, et al . Thrombocytopenia in patients with advanced liver disease. Hematology 2002;90:104-8.  Back to cited text no. 20    

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Correspondence Address:
Madiha M El-Attar
Department of Tropical Medicine and Gastroenterology, Faculty of Medicine, Assuit University, Assiut 71111
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1319-3767.32181

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