- Email: [email protected]
Thrombocytopenia associated with hepatitis C viral infection
Journal of Hepatology 1996;24: 135-140 Printed in Denmark . AN rights reserved
Copyrigkf 0 European Association for the Study of the Liver 1996 Journal of Hepatology ISSN 0168-8278
Thrombocytopenia associated with hepatitis C viral infixtion Takeaki Nagamine,
Tosiyuki Ohtuka,
Ken Takehara,
Takayuki
Arai, Hitoshi Takagi and Masatomo
Mori
First Department of Internal Medicine. Gunma University School of Medicine, Maebashi, Japan
Background/Aims: We investigated whether chronic hepatitis C infection is associated with thrombocytopenia. Thrombocytopenia (< 15 x lo4 Methods/Results: platelets/$) was diagnosed in 151 of 368 patients (41.0%) with chronic hepatitis C, a significantly higher proportion than that observed in patients with chronic hepatitis B (18.9%, ~~0.01). Elevated titers of platelet-associated imnmnoglobulin G were observed in 88.1% of patients with chronic hepatitis C and in 47.1% of patients with chronic hepatitis B. Platelet-associated imnmnoglobulin G titers were siguiflcantly higher in patients with chronic hepatitis C (87.3klO.l ng/lO’ cells) vs. those with chronic hepatitis B (30.326.4) or the control subjects (p
platelet-associated immunoglobulin G titers and platelet counts in both chronic hepatitis C and B cases. Platelet-associated immunoglobulin G titers were significantly higher in patients with type C cirrhosis than in those with type B cirrhosis. Interestingly, HCV-RNA was detected by RT-PCR in the platelets from 11 of 14 patients with hepatitis C virus. Conclusions: These data indicate that chronic infection with hepatitis C virus may produce a significant autoimmune reaction to platelets, leading to thrombocytopenia.
T
the production of platelet-associated immunoglobulin G (PAIgG) that binds to platelet-specific antigens (1 l13). We conducted the present study to investigate whether HCV infection reflects the expression of PAIgG, possibly leading to platelet destruction by the reticula-endothelial system. Since platelets have been found to be directly infected by viruses such as the human immunodeficiency virus (HIV) and herpes simplex virus (14,15), we also measured levels of HCVRNA in platelets obtained from patients with HCV
can be caused by an autoimmune reaction to platelets. This blood dyscrasia is associated with a variety of infections (1). Several reports have shown that thrombocytopenia is associated with acute hepatitis virus A and B infections (2,3). Graver et al. (4) reported that 6 of 16 patients with chronic viral hepatitis exhibited thrombocytopenia; however, the type of hepatitis virus was not indicated. Although some data exist, it is not known whether hepatitis C virus (HCV) is responsible for the thrombocytopenia (5). HCV infection is commonly associated with immune disturbances. These include the development of autoantibodies (6), an increased frequency of immune-mediated diseases such as mixed cryoglobulinemia, findings resembling those of Sjbgren’s syndrome, and membranoproliferative glomerulonephritis (7-10). Idiopathic thrombocytic purpura (ITP) is associated with HROMBOCYTOPENIA
Received 16 March; revised 17 July; accepted 24 July 1995
Correspondence: Takeaki Nagamine, M.D., First Department of Internal Medicine, Gunma University School of Medicine, Maebashi 371, Japan.
Key words: Chronic hepatitis C; HCV-RNA; Platelet-associated immunoglobulin G; Thrombocytopenia.
Materials
and Methods
Subjects and clinical data
Between January 1992 and December 1993, 421 patients with chronic viral hepatitis underwent liver biopsy at Gunma University School Hospital and its affiliated institutions. A second-generation enzymelinked immunosorbent assay (Ortho, Raritan, N.J., USA), using an antibody to hepatitis C virus was positive in 368 cases of chronic viral hepatitis. Hepatitis B surface antigen, as assayed by a passive hemagglutination test (Fuji Rebio, Tokyo, Japan), was positive in 53 135
T. Nagamine
et al.
cases. Liver function tests, and leukocyte and platelet counts were determined in the hospital laboratory by using an autoanalyzer at the time of liver biopsy. The results were compared between patients with chronic hepatitis B and hepatitis C (Table 1). Morphologic changes in the specimens of liver obtained from patients with chronic hepatitis were subdivided into chronic persistent hepatitis and chronic active hepatitis. The latter group was further qualified according to the histological guidelines of Sheuer as minimal or mild, moderate, and severe advanced active hepatitis (16). The degree of histological severity was similar in patients with chronic hepatitis B vs. those with chronic hepatitis C. The presence of cirrhosis and portal hypertension was obviated in all chronic hepatitis cases by physiological signs, results of laboratory tests, uitrasonography and/or computed tomography, and histological findings. Patients with chronic hepatitis C also exhibited various disorders of the immune system, such as Hashimoto’s thyroiditis (5 cases), rheumatoid arthritis (4 cases), bronchial asthma (3 cases), progressive systemic sclerosis (2 cases), Graves’ disease
(1 case), ulcerative colitis (1 case), sarcoidosis (1 case), and Sjogren syndrome (1 case). Patients with chronic hepatitis B exhibited Hashimoto’s thyroiditis (2 cases) and Vogt-Koyanagi-Harada syndrome (1 case). Other infections (HIV, herpes simplex, and others) that would affect the bone marrow, were not present in these patients. Seventeen patients with chronic hepatitis B (8 males and 9 females, mean age; mean-+SE=40.9+3.5) and 67 patients with chronic hepatitis C (32 males and 35 females, mean age; 49.7+ 1.3) were admitted to Gunma University School Hospital. Serving as controls were 49 healthy volunteers (31 males and 18 females, mean
PAlgG blg/lO cells
. 300
. TABLE
. . .
1
Characteristics
of the subiects Chronic hepatitis (n=53) 42.8kl.6
Age (years) Sex Male Female Duration
Chronic hepatitis (n=368)
of the disease (years)
Laboratory findings ALT (IU/l) T. bilirubin (mg/dl) Albumin (g/dl) y-globulin (g/dl) Leukocyte
count
(/PI)
*p
: .
200
. .. .
233 145
5.5kO.7
.
5.7kO.3
5
34
17 20 11
I51 129 54
. 100
.. . :. .. ..
. 99.4% 13.7 0.82t0.08 4.19?0.08 1.30~0.05
90.154.4 0.72+0.03 4.39zo.04 1.6220.04
55052222
5402?81
Platelet count (X 104/& (normal range: 1635) >20.0 15.0-19.9 10.0-14.9 <9.9 Range MeanrtSE
C
50.3k1.2
33 20
Histological classification Chronic persistent hepatitis Chronic active hepatitis Minimal or mild Moderate Severe
136
B
9.G32.0 19.3t0.7 compared
with chronic
. . . .
25.
97 120 117 34 5.5-34.0 16.7?0.3* hepatitis
..y’.n
. .
Control
23 20 9 I
::.
.
50.
B.
Chronic hepatitis
B
Chronic hepatitis
C
Fig. I. Platelet-associated immunoglobulin-G (PAIgG) levels in chronic viral hepatitis and healthy controls. Abnormally elevated PAIgG levels are observed in 88.1% of the chronic hepatitis C patients and in 47.1% of the chronic hepatitis B patients. PAIgG levels are significantly higher in patients with chronic hepatitis C relative to those with chronic hepatitis B. ** p
Thrombocytopenia PAlgG h&O cells)
= -0.32 40.05 100
1
I
SO-
01 0
.. .. -----_\i_\l r =
-0.59
P co.01
.
4’
l
.
5
10
15
20
25
30
Platelet count (low)
age; 44.5? 1.2). Blood samples were immediately collected for assay for PAIgG. The control subjects were negative for hepatitis B and C. In addition, serum PAIgG levels were assayed in eight patients with type B cirrhosis (5 males and 3 females, mean age; 56?3) and in 15 patients with type C cirrhosis (8 males and 7 females, mean age; 63 + 3). Informed consent was obtained from each subject prior to collection of blood samples. determination
PAIgG was determined by using a competitive microenzyme-linked immunosorbent assay (ELISA) as described by Kawaguchi et al. (17). In brief, platelets were separated from whole blood collected in EDTA, and washed with phosphate-buffered saline containing 1.l% bovine serum albumin. The ELISA was performed in 96-well microplates coated with purified human IgG. A horseradish peroxidase-conjugated antihuman IgG antibody was incubated simultaneously with the samples, and visualized with o-phenylenediamine.
TABLE
PAIgG levels (ng/lO’ cells) control
(n=49)
16.6kO.6
Histologic classification of chronic hepatitis C Chronic persistent hepatitis (n=8) 23.325.0 Chronic active hepatitis Minimal or mild (n=27) 69.0? 15.4* Moderate (n= 19) 110.1~18.3* Severe (n= 13) 121.8?27.3* * (J~0.01)
RNA
Fig. 2. Correlation between PAIgG titers and platelet counts. The titers of PAIgG show a signljicant negative correlation to platelet counts in patients with chronic hepatitis B (Right) and in those with chronic hepatitis C (Left).
determination
HCV-RNA in peripheral platelets was determined prospectively in 13 chronic hepatitis C patients, five chronic hepatitis B patients, and five healthy volunteers. Nine of 13 patients with chronic hepatitis C had both thrombocytopenia and elevated PAIgG levels, while four patients with chronic hepatitis C had both normal platelet counts and normal PAIgG levels. Platelets were separated from peripheral blood as follows. Ten milliliters of whole blood were collected into tubes containing 0.1 vol. of 3.85% sodium citrate and subsequently centrifuged at 150Xg for 10 min at room temperature. The platelet-rich plasma was then collected and acidified with 0.01 vol. of 1M citrate. After centrifugation at 1OOOXgfor 15 min, the platelet pellets were washed three times with Tyrode’s solution (137 mM NaCl, 0.42 mM NaH2P04, 5 mM glucose, 0.35% albumin, pH 7.35). Total RNA was obtained from the washed platelets by using the guanidium-isothiocyanate method. Reverse transcription (RT) of total platelet RNA (
2
Relationship between platelet-associated immunoglobulin-G levels and morphologic findings of chronic hepatitis C
Healthy
infection
PAlgG hg/W cells) 150
PAIgG
in HCV
Significant
difference
compared
(PAIgG) TABLE
Platelet counts (X104/pl)
3
Detection rate of HCV-RNA by polymerase chain reaction platelets from patients with chronic hepatitis C
23.5kl.l
No. of patients
Identification of HCV-RNA in platelets
Thrombocytopenia associated elevated level of PAIgG
9
9 (100%)
Normal platelet count normal PAIgG levels
4
2 (50%)
22.8k 1.2 18.0t0.9* 14.5+1.3* ll.OkO.S*
with healthy
control.
(PCR) in
and
137
T. Nagamine
et al.
the Tyrode’s solution used to wash the platelet pellets was used as a negative control. Statistical analysis Data are expressed as mean?SEM. Analysis of variance and chi-square tests were used to determine the significance of the differences between data from each patient group. A level of pcO.05 was accepted as statistically significant.
Results The incidence of thrombocytopenia (< 15 X lo4 platelets/pi) was significantly higher in patients with chronic hepatitis C (41 .O%) than in those with chronic hepatitis B (18.9%) (Table 1). The titers of PAIgG in control subjects and patients with hepatitis B and C are shown in Fig. 1. The PAIgG in control subjects (n=49) ranged from 9.0 to 25.0 ng/ lo7 cells. Abnormally elevated PAIgG levels were observed in 88.1% of the chronic hepatitis C patients and in 47.1% of the chronic hepatitis B patients. Mean PAIgG values were significantly higher in those with chronic hepatitis C (87.3210.1) than in those with chronic hepatitis B (30.326.4) or the control subjects (16.6kO.6). The mean values for the serum IgG levels (normal range: 770-1700 mg/dl) were 2153.0568.2 in chronic hepatitis C and 1641.65 110.5 in chronic hepatitis B. A significant negative correlation Q~0.05) between PAIgG and platelet counts was observed in patients with chronic hepatitis C (Fig. 2) but PAIgG levels did not correlate with serum IgG values. Similarly, patients with chronic hepatitis B exhibited a significant negative correlation QKO.01) between PAIgG levels and platelet counts. The relationship between the PAIgG levels and the histological findings seen during chronic hepatitis C is shown in Table 2. PAIgG levels increased with the degree of histological progression and caused a subsequent decrease in platelet counts. PAIgG levels were significantly higher (‘~~0.05) in patients with type C cirrhosis (284255) vs. type B cirrhosis (78~~9). Fig. 3 shows the presence of HCV-RNA in platelets isolated from peripheral blood of patients with chronic hepatitis C. HCV-RNA was detected in the platelets taken from 11 of 13 chronic hepatitis C patients. No HCV-RNA was detected in platelets taken from the chronic hepatitis B patients, healthy subjects, or in the final Tyrode’s solution used to separate platelets. HCVRNA was identified in platelets from all patients showing both thrombocytopenia and elevated PAIgG levels, as well as in two out of four patients with both normal platelet counts and PAIgG levels (Table 3).
138
Fig. 3. Analysis of HCV-RNA in platelet pellets by nested RT-PCR analysis. Platelets were separatedfrom peripheral blood ofpatients with types B and C chronic hepatitis. Total RNA was extractedfrom the platelet pellets and cDNA was synthesized. A two-step PCR assay, with nested primers, was performed. An amplication product at the expectedposition of 145 bp is seen only in patients with chronic hepatitis C. Lane I, ethidium bromide-stained size markers; Lane 2, cDNA from type B chronic hepatitis; Lane 3, cDNA from type C chronic hepatitis; Lane 4, the final washing solution as a negative control.
Discussion Our data clearly demonstrated a correlation between chronic infection with HCV and thrombocytopenia. We also demonstrated a significant elevation of PAIgG in patients with chronic hepatitis B or C compared with normal subjects. Patients with chronic hepatitis C had significantly higher PAIgG levels than patients with chronic hepatitis B. PAIgG titers were higher in patients with type C cirrhosis relative to those with type B cirrhosis. There was also a significant correlation between the titers of PAIgG and the severity and architectural changes in liver histology in patients with
Thrombocytopenia in HCV infection
chronic hepatitis C. Although previous observations showed that an elevation of PAIgG paralleled the severity of liver disease (4) the association between infections by differing viruses had not been demonstrated. The findings suggest that a prolonged infection with HCV causes pronounced immune dysfunction, resulting in high PAIgG titers. A significant inverse correlation between PAIgG levels and blood platelet counts was previously observed in ITP (12), and was corroborated by the present data. The autoantibodies thought to cause platelet destruction in ITP are those reactive against surface epitopes of the major platelet membrane glycoproteins (13). HCV infection is also associated with certain autoimmune diseases (7-10). In the present study, the presence of autoimmune diseases coincided more often with chronic hepatitis C than B. Although the underlying mechanisms for the autoimmunity require an identical amino acid sequence homology between bacteria and their protein components, it is not known whether there is a strong homology between the HCV proteins and platelet protein components (1,19). Despite previous reports describing a nonspecific adsorption of elevated IgG by platelets in chronic liver disease, we found no relationship between PAIgG and serum IgG values in patients with chronic hepatitis C (4,20). The technique used for quantitating PAIgG appears to measure total PAIgG, which is not equivalent to platelet-specific autoantibodies (17). Studies are in progress to identify the specific autoantibodies against platelet membrane glycoproteins in patients with chronic hepatitis C. Alternatively, a direct viral infection of the platelets may account for the thrombocytopenia. HIV has been demonstrated in platelets from patients with thrombocytopenia (14). This observation prompted us to investigate the existence of HCV in platelets from patients with chronic hepatitis C. Our data showed the presence of HCV-RNA in platelets isolated from these patients but not in patients with hepatitis B or control subjects. We believe that this RNA is derived from bound or adsorbed viral particles on the platelets (14,15). Silva et al. (5) reported the detection of HCV-RNA in platelets from patients with ITP-associated HCV infection, but not in those without ITP This suggests that HCV may be involved in the pathogenesis of ITP in some patients. Similarly, the present study showed that HCVRNA was identified in platelets from all patients with thrombocytopenia and elevated PAIgG levels. It is important to note that while HCV-RNA was identified in platelets from half of our patients with normal platelet counts and PAIgG levels, it is not known whether the bound or adsorbed HCV on the platelet surface re-
sulted in PAIgG expression. Interestingly, Hijikata et al. (21) showed that HCV bound to immunoglobulin in the peripheral blood and elicited immune disturbance. Antigen-antibody complexes may be another cause for platelet sensitization and thrombocytopenia.
References 1. Bithell TC. Miscellaneous
forms of thrombocytopenia. In: Lee GR, Bithell TC, Foerster J, Athens J, Lukens JN, eds. Wintrobe’s Clinical Hematology, 9th Edn. Philadelphia: Lea & Febiger, 1993; 1363-73. associated 2. Romero R, Kleinman RE. Thrombocytopenia with acute hepatitis B infection. Peditrics 1993; 39: 543-9. 3. Ibarra H, Zapata C, Inostroza J, Mezzano S, Riedemann S. Immune thrombocytopenic purpura associated with hepatitis A. Blut 1986; 52: 371-5. 4. Graver D, Giuliani D, Leevy CM, Morse BS. Platelet-associated IgG in hepatitis and cirrhosis. J Clin Immunol 1984; 4: 108-l 1. 5. Silva M, Li X, Cheinquer H, Kolodny L, Radick J, LaRue S, Hill M, de Medina M, Reddy R, Jeffers L, Roach K, Schiff ER. HCV associated idiopathic thrombocytopenic purpura (ITP). Gastroenterelogy 1992; 102: A889. 6. Pawlotsky JM, Yahia MB, Andre C, Voisin MC, Intrator L, Roudot, Roudot-Thoraval F, Deforges L, Duvoux C, Zafrani E, Duval J, Dhumeaux D. Immunological disorders in C virus chronic active hepatitis: a prospective case-control study. Hepatology 1994; 19: 841-8. 7. Pascual M, Perrin L, Giostra E, Schifferli JA. Hepatitis C virus in patients with cryoglobulinemia type II. J Infect Dis 1990; 162: 569-70. 8. Haddad J, Deny P, Munz-Gotheil C, Ambrosini JC, Trinchet JC, Paterson D, Ma1 F, Callard P Beaugrand M. Lymphocytic sialadenitis of Sjiigren’s syndrome associated with chronic hepatitis C virus liver disease. Lancet 1992; 339: 321-3. 9. Johnson RJ, Gretch DR, Yamabe H, Hart J, Bacchi CE, Hartwell P Couser WG, Corey L, Wener MH, Alpers E. Membranoproliferative glomerulonephritis associated with hepatitis C virus infection. N Engl J Med 1993; 328: 46570. 10. Lunel E HCV and autoimmunity: fortuitous association or reality? Gastroenterology 1994; 107: 1550-55. purpura. 11. McMillan R. Chronic idiopathic thrombocytopenic N Engl J Med 1981; 304: 113547. 12. Sixon R, Rosse W, Ebbert L. Quantitative determination of antibody in idiopathic thrombocytopenic purpura. N Engl J Med 1975; 292: 23&6. 13. Fujisawa K, Tani P, O’Toole TE, Ginsbery MH, McMillan R. Different specificities of platelet-associated and plasma autoantibodies to platelet GPIIb-IIIa in patients with chronic immune thrombocytopenic purpura. Blood 1992; 79: 14416. 14. Ballem PJ, Beizberg A, Devine DV, Lyster D, Spruston B, Chamber H, Doubroff PRT, Mikulash KMC. Kinetic studies on the mechanism of thrombocytopenia in patients with human immunodeficiency virus infection. N Engl J Med 1992; 327: 1779-84. 15 Forghani B, Schmidt NJ. Association of herpes simplex virus with platelets of experimentally infected mice. Arch Virol 1983; 76: 269-74. 16. Sheuer PJ. Liver Biopsy Interpretation, 3rd Edn. London: Bailliere Tindall, 1981.
139
T. Nagamine
et al.
17. Kawaguchi R, Haruna S, Yonezawa M, Hikichi K, Nomura T. Determination of human platelet associated IgG using a competitive micro ELISA method and its clinical applications. Jpn Clin Chem 1986; 15: 277-84. 18. Okamoto H, Okada S, Sugiyama Y, Tanaka T, Sugai Y, Akahane Y, Machida A, Mishiro S, Yoshizawa H, Miyakawa Y. Detection of hepatic C virus RNA by a two-stage polymerase chain reaction with two pairs of primers deduced from the 5-noncoding region. Jpn J Exp Med 1990; 60: 215-22. 19. Hohmann AW, Booth K, Peter SV, Gordon DL, Comacchio
140
RM. Common epitope on HIV p24 and human platelets. Lancet 1993; 342: 1274-5. 20. de Noronha R, Taylor BA, Wild G, Triger DR, Greaves M. Interrelationships between platelet count, platelet IgG, serum IgG, immune complexes and severity of liver disease. Clin Lab Haematol 1991; 13: 127-35. 21. Hijikata M, Shimizu Y, Kato H, Iwamoto A, James WS, Alter HJ, Purcell RH, Yoshikura H. Equilibrium centrifugation studies of hepatitis C virus: evidence for circulating immune complexes. J Virol 1993; 67: 1953-8.
Copyrigkf 0 European Association for the Study of the Liver 1996 Journal of Hepatology ISSN 0168-8278
Thrombocytopenia associated with hepatitis C viral infixtion Takeaki Nagamine,
Tosiyuki Ohtuka,
Ken Takehara,
Takayuki
Arai, Hitoshi Takagi and Masatomo
Mori
First Department of Internal Medicine. Gunma University School of Medicine, Maebashi, Japan
Background/Aims: We investigated whether chronic hepatitis C infection is associated with thrombocytopenia. Thrombocytopenia (< 15 x lo4 Methods/Results: platelets/$) was diagnosed in 151 of 368 patients (41.0%) with chronic hepatitis C, a significantly higher proportion than that observed in patients with chronic hepatitis B (18.9%, ~~0.01). Elevated titers of platelet-associated imnmnoglobulin G were observed in 88.1% of patients with chronic hepatitis C and in 47.1% of patients with chronic hepatitis B. Platelet-associated imnmnoglobulin G titers were siguiflcantly higher in patients with chronic hepatitis C (87.3klO.l ng/lO’ cells) vs. those with chronic hepatitis B (30.326.4) or the control subjects (p
platelet-associated immunoglobulin G titers and platelet counts in both chronic hepatitis C and B cases. Platelet-associated immunoglobulin G titers were significantly higher in patients with type C cirrhosis than in those with type B cirrhosis. Interestingly, HCV-RNA was detected by RT-PCR in the platelets from 11 of 14 patients with hepatitis C virus. Conclusions: These data indicate that chronic infection with hepatitis C virus may produce a significant autoimmune reaction to platelets, leading to thrombocytopenia.
T
the production of platelet-associated immunoglobulin G (PAIgG) that binds to platelet-specific antigens (1 l13). We conducted the present study to investigate whether HCV infection reflects the expression of PAIgG, possibly leading to platelet destruction by the reticula-endothelial system. Since platelets have been found to be directly infected by viruses such as the human immunodeficiency virus (HIV) and herpes simplex virus (14,15), we also measured levels of HCVRNA in platelets obtained from patients with HCV
can be caused by an autoimmune reaction to platelets. This blood dyscrasia is associated with a variety of infections (1). Several reports have shown that thrombocytopenia is associated with acute hepatitis virus A and B infections (2,3). Graver et al. (4) reported that 6 of 16 patients with chronic viral hepatitis exhibited thrombocytopenia; however, the type of hepatitis virus was not indicated. Although some data exist, it is not known whether hepatitis C virus (HCV) is responsible for the thrombocytopenia (5). HCV infection is commonly associated with immune disturbances. These include the development of autoantibodies (6), an increased frequency of immune-mediated diseases such as mixed cryoglobulinemia, findings resembling those of Sjbgren’s syndrome, and membranoproliferative glomerulonephritis (7-10). Idiopathic thrombocytic purpura (ITP) is associated with HROMBOCYTOPENIA
Received 16 March; revised 17 July; accepted 24 July 1995
Correspondence: Takeaki Nagamine, M.D., First Department of Internal Medicine, Gunma University School of Medicine, Maebashi 371, Japan.
Key words: Chronic hepatitis C; HCV-RNA; Platelet-associated immunoglobulin G; Thrombocytopenia.
Materials
and Methods
Subjects and clinical data
Between January 1992 and December 1993, 421 patients with chronic viral hepatitis underwent liver biopsy at Gunma University School Hospital and its affiliated institutions. A second-generation enzymelinked immunosorbent assay (Ortho, Raritan, N.J., USA), using an antibody to hepatitis C virus was positive in 368 cases of chronic viral hepatitis. Hepatitis B surface antigen, as assayed by a passive hemagglutination test (Fuji Rebio, Tokyo, Japan), was positive in 53 135
T. Nagamine
et al.
cases. Liver function tests, and leukocyte and platelet counts were determined in the hospital laboratory by using an autoanalyzer at the time of liver biopsy. The results were compared between patients with chronic hepatitis B and hepatitis C (Table 1). Morphologic changes in the specimens of liver obtained from patients with chronic hepatitis were subdivided into chronic persistent hepatitis and chronic active hepatitis. The latter group was further qualified according to the histological guidelines of Sheuer as minimal or mild, moderate, and severe advanced active hepatitis (16). The degree of histological severity was similar in patients with chronic hepatitis B vs. those with chronic hepatitis C. The presence of cirrhosis and portal hypertension was obviated in all chronic hepatitis cases by physiological signs, results of laboratory tests, uitrasonography and/or computed tomography, and histological findings. Patients with chronic hepatitis C also exhibited various disorders of the immune system, such as Hashimoto’s thyroiditis (5 cases), rheumatoid arthritis (4 cases), bronchial asthma (3 cases), progressive systemic sclerosis (2 cases), Graves’ disease
(1 case), ulcerative colitis (1 case), sarcoidosis (1 case), and Sjogren syndrome (1 case). Patients with chronic hepatitis B exhibited Hashimoto’s thyroiditis (2 cases) and Vogt-Koyanagi-Harada syndrome (1 case). Other infections (HIV, herpes simplex, and others) that would affect the bone marrow, were not present in these patients. Seventeen patients with chronic hepatitis B (8 males and 9 females, mean age; mean-+SE=40.9+3.5) and 67 patients with chronic hepatitis C (32 males and 35 females, mean age; 49.7+ 1.3) were admitted to Gunma University School Hospital. Serving as controls were 49 healthy volunteers (31 males and 18 females, mean
PAlgG blg/lO cells
. 300
. TABLE
. . .
1
Characteristics
of the subiects Chronic hepatitis (n=53) 42.8kl.6
Age (years) Sex Male Female Duration
Chronic hepatitis (n=368)
of the disease (years)
Laboratory findings ALT (IU/l) T. bilirubin (mg/dl) Albumin (g/dl) y-globulin (g/dl) Leukocyte
count
(/PI)
*p
: .
200
. .. .
233 145
5.5kO.7
.
5.7kO.3
5
34
17 20 11
I51 129 54
. 100
.. . :. .. ..
. 99.4% 13.7 0.82t0.08 4.19?0.08 1.30~0.05
90.154.4 0.72+0.03 4.39zo.04 1.6220.04
55052222
5402?81
Platelet count (X 104/& (normal range: 1635) >20.0 15.0-19.9 10.0-14.9 <9.9 Range MeanrtSE
C
50.3k1.2
33 20
Histological classification Chronic persistent hepatitis Chronic active hepatitis Minimal or mild Moderate Severe
136
B
9.G32.0 19.3t0.7 compared
with chronic
. . . .
25.
97 120 117 34 5.5-34.0 16.7?0.3* hepatitis
..y’.n
. .
Control
23 20 9 I
::.
.
50.
B.
Chronic hepatitis
B
Chronic hepatitis
C
Fig. I. Platelet-associated immunoglobulin-G (PAIgG) levels in chronic viral hepatitis and healthy controls. Abnormally elevated PAIgG levels are observed in 88.1% of the chronic hepatitis C patients and in 47.1% of the chronic hepatitis B patients. PAIgG levels are significantly higher in patients with chronic hepatitis C relative to those with chronic hepatitis B. ** p
Thrombocytopenia PAlgG h&O cells)
= -0.32 40.05 100
1
I
SO-
01 0
.. .. -----_\i_\l r =
-0.59
P co.01
.
4’
l
.
5
10
15
20
25
30
Platelet count (low)
age; 44.5? 1.2). Blood samples were immediately collected for assay for PAIgG. The control subjects were negative for hepatitis B and C. In addition, serum PAIgG levels were assayed in eight patients with type B cirrhosis (5 males and 3 females, mean age; 56?3) and in 15 patients with type C cirrhosis (8 males and 7 females, mean age; 63 + 3). Informed consent was obtained from each subject prior to collection of blood samples. determination
PAIgG was determined by using a competitive microenzyme-linked immunosorbent assay (ELISA) as described by Kawaguchi et al. (17). In brief, platelets were separated from whole blood collected in EDTA, and washed with phosphate-buffered saline containing 1.l% bovine serum albumin. The ELISA was performed in 96-well microplates coated with purified human IgG. A horseradish peroxidase-conjugated antihuman IgG antibody was incubated simultaneously with the samples, and visualized with o-phenylenediamine.
TABLE
PAIgG levels (ng/lO’ cells) control
(n=49)
16.6kO.6
Histologic classification of chronic hepatitis C Chronic persistent hepatitis (n=8) 23.325.0 Chronic active hepatitis Minimal or mild (n=27) 69.0? 15.4* Moderate (n= 19) 110.1~18.3* Severe (n= 13) 121.8?27.3* * (J~0.01)
RNA
Fig. 2. Correlation between PAIgG titers and platelet counts. The titers of PAIgG show a signljicant negative correlation to platelet counts in patients with chronic hepatitis B (Right) and in those with chronic hepatitis C (Left).
determination
HCV-RNA in peripheral platelets was determined prospectively in 13 chronic hepatitis C patients, five chronic hepatitis B patients, and five healthy volunteers. Nine of 13 patients with chronic hepatitis C had both thrombocytopenia and elevated PAIgG levels, while four patients with chronic hepatitis C had both normal platelet counts and normal PAIgG levels. Platelets were separated from peripheral blood as follows. Ten milliliters of whole blood were collected into tubes containing 0.1 vol. of 3.85% sodium citrate and subsequently centrifuged at 150Xg for 10 min at room temperature. The platelet-rich plasma was then collected and acidified with 0.01 vol. of 1M citrate. After centrifugation at 1OOOXgfor 15 min, the platelet pellets were washed three times with Tyrode’s solution (137 mM NaCl, 0.42 mM NaH2P04, 5 mM glucose, 0.35% albumin, pH 7.35). Total RNA was obtained from the washed platelets by using the guanidium-isothiocyanate method. Reverse transcription (RT) of total platelet RNA (
2
Relationship between platelet-associated immunoglobulin-G levels and morphologic findings of chronic hepatitis C
Healthy
infection
PAlgG hg/W cells) 150
PAIgG
in HCV
Significant
difference
compared
(PAIgG) TABLE
Platelet counts (X104/pl)
3
Detection rate of HCV-RNA by polymerase chain reaction platelets from patients with chronic hepatitis C
23.5kl.l
No. of patients
Identification of HCV-RNA in platelets
Thrombocytopenia associated elevated level of PAIgG
9
9 (100%)
Normal platelet count normal PAIgG levels
4
2 (50%)
22.8k 1.2 18.0t0.9* 14.5+1.3* ll.OkO.S*
with healthy
control.
(PCR) in
and
137
T. Nagamine
et al.
the Tyrode’s solution used to wash the platelet pellets was used as a negative control. Statistical analysis Data are expressed as mean?SEM. Analysis of variance and chi-square tests were used to determine the significance of the differences between data from each patient group. A level of pcO.05 was accepted as statistically significant.
Results The incidence of thrombocytopenia (< 15 X lo4 platelets/pi) was significantly higher in patients with chronic hepatitis C (41 .O%) than in those with chronic hepatitis B (18.9%) (Table 1). The titers of PAIgG in control subjects and patients with hepatitis B and C are shown in Fig. 1. The PAIgG in control subjects (n=49) ranged from 9.0 to 25.0 ng/ lo7 cells. Abnormally elevated PAIgG levels were observed in 88.1% of the chronic hepatitis C patients and in 47.1% of the chronic hepatitis B patients. Mean PAIgG values were significantly higher in those with chronic hepatitis C (87.3210.1) than in those with chronic hepatitis B (30.326.4) or the control subjects (16.6kO.6). The mean values for the serum IgG levels (normal range: 770-1700 mg/dl) were 2153.0568.2 in chronic hepatitis C and 1641.65 110.5 in chronic hepatitis B. A significant negative correlation Q~0.05) between PAIgG and platelet counts was observed in patients with chronic hepatitis C (Fig. 2) but PAIgG levels did not correlate with serum IgG values. Similarly, patients with chronic hepatitis B exhibited a significant negative correlation QKO.01) between PAIgG levels and platelet counts. The relationship between the PAIgG levels and the histological findings seen during chronic hepatitis C is shown in Table 2. PAIgG levels increased with the degree of histological progression and caused a subsequent decrease in platelet counts. PAIgG levels were significantly higher (‘~~0.05) in patients with type C cirrhosis (284255) vs. type B cirrhosis (78~~9). Fig. 3 shows the presence of HCV-RNA in platelets isolated from peripheral blood of patients with chronic hepatitis C. HCV-RNA was detected in the platelets taken from 11 of 13 chronic hepatitis C patients. No HCV-RNA was detected in platelets taken from the chronic hepatitis B patients, healthy subjects, or in the final Tyrode’s solution used to separate platelets. HCVRNA was identified in platelets from all patients showing both thrombocytopenia and elevated PAIgG levels, as well as in two out of four patients with both normal platelet counts and PAIgG levels (Table 3).
138
Fig. 3. Analysis of HCV-RNA in platelet pellets by nested RT-PCR analysis. Platelets were separatedfrom peripheral blood ofpatients with types B and C chronic hepatitis. Total RNA was extractedfrom the platelet pellets and cDNA was synthesized. A two-step PCR assay, with nested primers, was performed. An amplication product at the expectedposition of 145 bp is seen only in patients with chronic hepatitis C. Lane I, ethidium bromide-stained size markers; Lane 2, cDNA from type B chronic hepatitis; Lane 3, cDNA from type C chronic hepatitis; Lane 4, the final washing solution as a negative control.
Discussion Our data clearly demonstrated a correlation between chronic infection with HCV and thrombocytopenia. We also demonstrated a significant elevation of PAIgG in patients with chronic hepatitis B or C compared with normal subjects. Patients with chronic hepatitis C had significantly higher PAIgG levels than patients with chronic hepatitis B. PAIgG titers were higher in patients with type C cirrhosis relative to those with type B cirrhosis. There was also a significant correlation between the titers of PAIgG and the severity and architectural changes in liver histology in patients with
Thrombocytopenia in HCV infection
chronic hepatitis C. Although previous observations showed that an elevation of PAIgG paralleled the severity of liver disease (4) the association between infections by differing viruses had not been demonstrated. The findings suggest that a prolonged infection with HCV causes pronounced immune dysfunction, resulting in high PAIgG titers. A significant inverse correlation between PAIgG levels and blood platelet counts was previously observed in ITP (12), and was corroborated by the present data. The autoantibodies thought to cause platelet destruction in ITP are those reactive against surface epitopes of the major platelet membrane glycoproteins (13). HCV infection is also associated with certain autoimmune diseases (7-10). In the present study, the presence of autoimmune diseases coincided more often with chronic hepatitis C than B. Although the underlying mechanisms for the autoimmunity require an identical amino acid sequence homology between bacteria and their protein components, it is not known whether there is a strong homology between the HCV proteins and platelet protein components (1,19). Despite previous reports describing a nonspecific adsorption of elevated IgG by platelets in chronic liver disease, we found no relationship between PAIgG and serum IgG values in patients with chronic hepatitis C (4,20). The technique used for quantitating PAIgG appears to measure total PAIgG, which is not equivalent to platelet-specific autoantibodies (17). Studies are in progress to identify the specific autoantibodies against platelet membrane glycoproteins in patients with chronic hepatitis C. Alternatively, a direct viral infection of the platelets may account for the thrombocytopenia. HIV has been demonstrated in platelets from patients with thrombocytopenia (14). This observation prompted us to investigate the existence of HCV in platelets from patients with chronic hepatitis C. Our data showed the presence of HCV-RNA in platelets isolated from these patients but not in patients with hepatitis B or control subjects. We believe that this RNA is derived from bound or adsorbed viral particles on the platelets (14,15). Silva et al. (5) reported the detection of HCV-RNA in platelets from patients with ITP-associated HCV infection, but not in those without ITP This suggests that HCV may be involved in the pathogenesis of ITP in some patients. Similarly, the present study showed that HCVRNA was identified in platelets from all patients with thrombocytopenia and elevated PAIgG levels. It is important to note that while HCV-RNA was identified in platelets from half of our patients with normal platelet counts and PAIgG levels, it is not known whether the bound or adsorbed HCV on the platelet surface re-
sulted in PAIgG expression. Interestingly, Hijikata et al. (21) showed that HCV bound to immunoglobulin in the peripheral blood and elicited immune disturbance. Antigen-antibody complexes may be another cause for platelet sensitization and thrombocytopenia.
References 1. Bithell TC. Miscellaneous
forms of thrombocytopenia. In: Lee GR, Bithell TC, Foerster J, Athens J, Lukens JN, eds. Wintrobe’s Clinical Hematology, 9th Edn. Philadelphia: Lea & Febiger, 1993; 1363-73. associated 2. Romero R, Kleinman RE. Thrombocytopenia with acute hepatitis B infection. Peditrics 1993; 39: 543-9. 3. Ibarra H, Zapata C, Inostroza J, Mezzano S, Riedemann S. Immune thrombocytopenic purpura associated with hepatitis A. Blut 1986; 52: 371-5. 4. Graver D, Giuliani D, Leevy CM, Morse BS. Platelet-associated IgG in hepatitis and cirrhosis. J Clin Immunol 1984; 4: 108-l 1. 5. Silva M, Li X, Cheinquer H, Kolodny L, Radick J, LaRue S, Hill M, de Medina M, Reddy R, Jeffers L, Roach K, Schiff ER. HCV associated idiopathic thrombocytopenic purpura (ITP). Gastroenterelogy 1992; 102: A889. 6. Pawlotsky JM, Yahia MB, Andre C, Voisin MC, Intrator L, Roudot, Roudot-Thoraval F, Deforges L, Duvoux C, Zafrani E, Duval J, Dhumeaux D. Immunological disorders in C virus chronic active hepatitis: a prospective case-control study. Hepatology 1994; 19: 841-8. 7. Pascual M, Perrin L, Giostra E, Schifferli JA. Hepatitis C virus in patients with cryoglobulinemia type II. J Infect Dis 1990; 162: 569-70. 8. Haddad J, Deny P, Munz-Gotheil C, Ambrosini JC, Trinchet JC, Paterson D, Ma1 F, Callard P Beaugrand M. Lymphocytic sialadenitis of Sjiigren’s syndrome associated with chronic hepatitis C virus liver disease. Lancet 1992; 339: 321-3. 9. Johnson RJ, Gretch DR, Yamabe H, Hart J, Bacchi CE, Hartwell P Couser WG, Corey L, Wener MH, Alpers E. Membranoproliferative glomerulonephritis associated with hepatitis C virus infection. N Engl J Med 1993; 328: 46570. 10. Lunel E HCV and autoimmunity: fortuitous association or reality? Gastroenterology 1994; 107: 1550-55. purpura. 11. McMillan R. Chronic idiopathic thrombocytopenic N Engl J Med 1981; 304: 113547. 12. Sixon R, Rosse W, Ebbert L. Quantitative determination of antibody in idiopathic thrombocytopenic purpura. N Engl J Med 1975; 292: 23&6. 13. Fujisawa K, Tani P, O’Toole TE, Ginsbery MH, McMillan R. Different specificities of platelet-associated and plasma autoantibodies to platelet GPIIb-IIIa in patients with chronic immune thrombocytopenic purpura. Blood 1992; 79: 14416. 14. Ballem PJ, Beizberg A, Devine DV, Lyster D, Spruston B, Chamber H, Doubroff PRT, Mikulash KMC. Kinetic studies on the mechanism of thrombocytopenia in patients with human immunodeficiency virus infection. N Engl J Med 1992; 327: 1779-84. 15 Forghani B, Schmidt NJ. Association of herpes simplex virus with platelets of experimentally infected mice. Arch Virol 1983; 76: 269-74. 16. Sheuer PJ. Liver Biopsy Interpretation, 3rd Edn. London: Bailliere Tindall, 1981.
139
T. Nagamine
et al.
17. Kawaguchi R, Haruna S, Yonezawa M, Hikichi K, Nomura T. Determination of human platelet associated IgG using a competitive micro ELISA method and its clinical applications. Jpn Clin Chem 1986; 15: 277-84. 18. Okamoto H, Okada S, Sugiyama Y, Tanaka T, Sugai Y, Akahane Y, Machida A, Mishiro S, Yoshizawa H, Miyakawa Y. Detection of hepatic C virus RNA by a two-stage polymerase chain reaction with two pairs of primers deduced from the 5-noncoding region. Jpn J Exp Med 1990; 60: 215-22. 19. Hohmann AW, Booth K, Peter SV, Gordon DL, Comacchio
140
RM. Common epitope on HIV p24 and human platelets. Lancet 1993; 342: 1274-5. 20. de Noronha R, Taylor BA, Wild G, Triger DR, Greaves M. Interrelationships between platelet count, platelet IgG, serum IgG, immune complexes and severity of liver disease. Clin Lab Haematol 1991; 13: 127-35. 21. Hijikata M, Shimizu Y, Kato H, Iwamoto A, James WS, Alter HJ, Purcell RH, Yoshikura H. Equilibrium centrifugation studies of hepatitis C virus: evidence for circulating immune complexes. J Virol 1993; 67: 1953-8.