High Prevalence of Serum Cryoglobulins in Multitransfused Hemophilic Patients With Chronic Hepatitis C

SERUM CRYOGLOBULINS, consisting of polyclonal IgG and either polyclonal or monoclonal (IgM) rheumatoid factor (mixed cryoglobulinemia) have been detected in 19% to 54% of patients with chronic hepatitis C virus (HCV) infection.1-5In 10% to 42% of the cases cryoglobulins were associated with a systemic vasculitic disorder with such clinical signs as palpable purpura, arthropathy, peripheral neuropathy, or Raynaud phenomenon.2,6 The prevalence, clinical relevance, and risk factors of serum cryoglobulins in patients with chronic hepatitis C are not well established. Cross-sectional studies of HCV-infected patients showed a predominance of serum cryoglobulins in women, older patients, and patients with long-lasting and severe hepatitis.6-8Other studies found a correlation between serum cryoglobulins and infection with HCV genotype 2a/c,9 genotype 1b,10 or any genotype.11 Studying serum cryoglobulins in hemophilic patients with chronic hepatitis C may be clinically important because the prevalence and clinical significance of cryoglobulins in these patients are unknown. Moreover, multitransfused hemophiliacs offer a unique model to evaluate the relationship between cryoglobulins and the duration of hepatitis C. The duration of hepatitis C can be established precisely in these patients, because they have been exposed to HCV at the time of their first infusion with non–virus-inactivated clotting factor concentrates.12-14 Therefore, we chose to study 135 multitransfused hemophilic patients with chronic hepatitis C who were consecutively recruited in this study during their periodic medical check-up at the Hemophilia Center (Milan, Italy).

From June 1995 to July 1996, we studied 135 consecutive anti-HCV antibody–positive hemophiliacs (134 men; 123 with hemophilia A and 12 with hemophilia B), who were regularly attending the Angelo Bianchi Bonomi Hemophilia and Thrombosis Center (Milan, Italy) for their annual medical check-up. The duration of HCV infection was calculated assuming that the first infusion with blood, plasma, or non–virus-inactivated concentrates manufactured from large pools of plasma had transmitted the virus C.12,13 According to this criterion, 128 hemophiliacs have been infected with HCV for 10 to 41 years (median, 23 years). Their main epidemiological and clinical features are shown in Table 1. In each patient serum alanine aminotransferase (ALT) values had been measured every year since 1979 and were classified as persistently normal (<40 U/L), intermittently elevated, and persistently elevated. HCV genotype distribution was 1a in 39%, 1b in 35%, 1a plus 1b in 3%, 2b in 4%, 2a/c in 2%, 3 in 10%, 4 in 2%, and not performed in 5%. Sixty-seven hemophiliacs (50%) had serum antibody to human immunodeficiency virus (anti-HIV) for 11 to 14 years (median, 13 years); 35 (52%) had signs of disease progression, with CD₄ cell counts less than 200/cmm and/or full-blown acquired immunodeficiency syndrome. These patients were receiving antiretroviral therapy. None of the patients had received treatment with interferon or steroids in the previous 12 months.

Clinical history and physical examination, liver and kidney function, and routine chemistry tests were obtained in all patients. Special attention was given to the search for signs and symptoms of cirrhosis and for cryoglobulinemic syndrome. Cirrhosis was diagnosed clinically on the basis of laboratory signs of liver failure, ie, platelet count less than 150,000/mL, serum albumin less than 3.5 g/L, serum cholinesterase activity less than 4,500 U/L, endoscopic signs of portal hypertension (presence of esophageal varices and hypertensive gastropathy), and/or by abdominal ultrasound (irregular margins of the liver, dilated portal vein, and splenomegaly). All patients were also examined for signs and symptoms referable to serum cryoglobulins, ie, palpable purpura, active or healed skin ulcers, peripheral neuropathy, Raynaud's phenomenon, sicca syndrome, or renal complications. Joint pain or damage were not considered among possible symptoms of cryoglobulinemia because hemophilic arthropathy may be a confounder.

Cryoglobulins were precipitated from serum stored for up to 8 days at 4°C (0.1 g/L sodium azide). The precipitates were washed five times at 4°C with 0.15 mol/L NaCl. A fraction of washed cryoglobulins was then diluted in 0.1 mol/L NaOH, and concentration was measured by reading the absorbance at 280 nm. Values less than 50 mg/L were considered negative. Washed cryoglobulins were tested for Ig composition by immunoblotting according to Musset et al.15According to Brouet et al,16 cryoglobulins were classified as type II (if rheumatoid factor was monoclonal) or type III (if rheumatology factor was polyclonal). Serum antinuclear antibodies, antimitochondrial antibodies, antismooth muscle antibodies, and anti–liver-kidney-microsome antibodies were tested for by indirect immunofluorescence. A titer equal or greater than 1:40 was considered positive. The serum Ig, rheumatoid factor, and C₃ and C₄ complement fraction levels were measured by routine nephelometric assays. The upper limit of the normal levels of the rheumatoid factor was 60 IU/mL.

Anti-HCV was detected with a third-generation enzyme-linked immunosorbent assay (ELISA; Ortho Diagnostics System, Raritan, NJ). HCV-RNA was detected by a polymerase chain reaction (PCR) technique (Amplicor, Roche Diagnostics, Nutley, NJ). HCV genotypes were determined by a reverse hybridization assay (LiPA; Innogenetics, Brussels, Belgium).17 Hepatitis B virus (HBV)-related markers were detected by using commercially available kits (Abbott Laboratories, North Chicago, IL). HBV-DNA was detected by a capture hybridization assay (Digene, Beltsville, MD). Anti–HIV-1 was detected by using a commercially available ELISA and confirmed by Western blot (Abbott Laboratories).

Continuous variables were expressed as mean values and standard deviations. Discrete variables were expressed as median values and ranges. Categorical values were expressed as frequency and percent values. The t-test was used to compare means. Logarithmic transformation was used when distribution was not normal. Logistic regression was used to calculate the risk of cryoglobulinemia in patients with serum HCV-RNA, HIV coinfection, and different duration of HCV infection. The periods of HCV infection duration were chosen on the basis of the distribution quartiles of the cohort (10 to 17, 18 to 21, 22 to 26, and greater than 26 years), and the first quartile was used as reference. The results were expressed as odds ratio and 95% confidence intervals (CI).

Forty-five patients (31%) circulated cryoglobulins, with concentrations ranging between 66 and 480 mg/L (median, 166 mg/L). Cryoglobulins were type III in 26 patients (62%) and type II in 12 (29%). In 4 patients (9%) cryoglobulins were not typed. Although in patients with type-II cryoglobulin there was a tendency for less severe liver disease (as expressed by the prevalence of persistently elevated ALT), differences were not statistically significant (35% v50% in type III). Age, severity of hemophilia, pattern of serum ALT, prevalence of cirrhosis, antitissue antibodies, anti-HCV, HBsAg, HBV-DNA, anti-HIV, and HIV disease progression were similar in patients with or without serum cryoglobulins (Table 1 and 2). There were no differences in HCV genotype distribution between cryoglobulinemic and noncryoglobulinemic patients (data not shown). Conversely, prevalence of serum HCV-RNA (95% v 80%, P < .05) and of rheumatoid factor (20%v 6%, P < .05), mean serum levels of IgG (2,354 ± 682 mg/dL v 1,928 ± 557 mg/dL, P < .0005), and IgM (323 ± 226 mg/dL v 244 ± 243 mg/dL, P< .05) were higher in patients with cryoglobulins than in those without. Instead, the mean serum levels of C₄ were lower in the cryoglobulinemic patients (19 ± 8 mg/dL v 24 ± 8 mg/dL, P < .05; Table 2). None of the patients had or has had signs of palpable purpura, healed or active skin ulcers, peripheral neuropathy, kidney failure, sicca syndrome, or Raynaud phenomenon. In patients with serum HCV-RNA the risk of developing serum cryoglobulins was 4.9 (95% CI, 1.1 to 22) times that for nonviremic patients and it remained significantly higher after adjusting for HIV infection (Table 3). Compared with patients with 10 to 17 years exposure to HCV, the risk of developing cryoglobulins was 2.4 (95% CI, 0.6 to 10.8) for patients with 18 to 21 years exposure to HCV, 3.7 (95% CI, 1.0 to 14.4) for those with 22 to 26 years exposure, and 4.4 (95% CI, 1.1 to 18.0) for those with longer than 26 years exposure. A similar trend was observed after adjusting for HIV infection. The odds ratio of developing serum cryoglobulins in anti-HIV–positive patients was 1.6 (95% CI, 0.8 to 3.2) compared with anti-HIV–seronegative patients (Table 3). Serum levels of cryoglobulins and type of cryoglobulin distribution were similar in anti-HIV–positive and –negative patients (data not shown). Anti-HIV–positive patients had higher serum levels of IgG (2,406 ± 608 mg/dL v 1,720 ± 435 mg/dL, P < .001) and IgM (295 ± 208 mg/dL v 243 ± 267 mg/dL, P < .05) than anti-HIV–seronegative patients.

This is the first study evaluating the prevalence, clinical relevance, and risk factors of serum cryoglobulins in multitransfused hemophilic patients with chronic HCV infection. Approximately one third of the patients circulated serum cryoglobulins, which were predominantly type III with polyclonal rheumatoid factor. Further confirming the strict association between HCV replication and appearance of serum cryoglobulins, serum HCV-RNA was more often detected in patients with cryoglobulins than in those without (95% v 80%, P < .05). Interestingly, despite the relatively high serum levels of circulating cryoglobulins, none of the patients had clinical signs or symptoms of systemic vasculitic disorders.

Three previous studies in nonhemophilic patients with HCV-related chronic hepatitis or cirrhosis found similar rates (35% to 37%) of serum cryoglobulins.2-4 Another study reported a 54% rate in patients with chronic hepatitis C.1 These studies showed a tendency for higher rates to occur in women, older patients, and those with long-lasting HCV-related infection or cirrhosis. Perhaps the fact that all except one of our hemophilic patients were men and had a median age of 31 years, and that only few of them had clinical signs of cirrhosis, would account for the relatively low rate (31%) of serum cryoglobulins that we observed. Studies in nonhemophilic patients suggested an association between serum cryoglobulins and genotype 2a/c or 1b of HCV.9,10 We could not assess whether any HCV genotype was pathogenetically relevant as to cryoglobulinemia, because most of our patients were chronically infected with the coagulation concentrate-related genotype 1 of HCV.18-20The low rates of genotype 2a/c could be another factor contributing to the relatively low rates of serum cryoglobulin found in these patients. Finally, it must be pointed out that this study was not prospective and was based on the evaluation of one serum sample only. It is possible that cryoglobulins may have appeared and disappeared over time in hemophilic patients with hepatitis C.

The most important finding of this study was the positive correlation existing between duration of hepatitis C and risk of producing serum cryoglobulins. The existence of such a correlation is difficult to show in nonhemophilic patients, because most of them have community-acquired hepatitis in which the starting point of infection is difficult to assess. Another important finding was the absence of clinical signs of systemic vasculitis in hemophiliacs with cryoglobulins. These data contrast with those obtained in nonhemophilic patients, showing signs and symptoms of vasculitis in 10% to 50% of patients.2,6,20,21 Perhaps such factors as the young age of our patients and the male sex, which seem to influence negatively the production of serum cryoglobulins, are also influencing the risk of vasculitis. Preliminary data indicate that patients infected with HCV later in life are at higher risk of cirrhosis and liver cancer than those infected earlier.22-24 Assuming that this holds true also for the risk of developing serum cryoglobulin-related sequelae, hemophiliacs could be at low risk of vasculitis because they were predominantly infected by HCV early in life at the time of the first infusion with blood products.

Similar prevalences and amounts of serum cryoglobulins were found in patients with serum anti-HIV and those without, indicating that there is no obvious association between HIV infection and cryoglobulins. However, association studies in HIV-infected hemophilic patients should be interpreted with caution. HIV/HCV-coinfected hemophiliacs could have more serious HCV-related sequelae than HIV-uninfected patients, but this is difficult to show by retrospective studies because of the shortened survival of patients with serum anti-HIV.25,26 

In conclusion, a significant number of hemophilic patients with chronic hepatitis C circulated cryoglobulins, but they had no clinical sign of vasculitis. Because the risk of developing serum cryoglobulins seems to increase with the duration of exposure to HCV, a prospective study is necessary to establish whether or not in these patients serum cryoglobulinemia will remain asymptomatic or become a clinically relevant complication of HCV infection.