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Cryofibrinogen in Patients with Hepatitis C Virus Infection
CLINICAL RESEARCH STUDY
Cryofibrinogen in Patients with Hepatitis C Virus Infection Aurélien Delluc, MD,a David Saadoun, MD, PhD,a Pascale Ghillani-Dalbin, PhD,b Damien Sene, MD, PhD,a Jean Charles Piette, MD,a Patrice Cacoub, MD, PhDa a
Service de Médecine Interne, Université Pierre et Marie Curie, Paris 6, France; bLaboratoire d’Immunochimie, Hôpital Pitié-Salpêtrière, Paris, France.
ABSTRACT BACKGROUND: Mixed cryoglobulin is usually associated with hepatitis C virus (HCV) infection and might cause systemic vasculitis. The presence and impact of cryofibrinogen, another cryoprotein, in the serum of HCV-infected patients have not yet been evaluated. The objective was to study the prevalence and the clinical and therapeutic impacts of cryofibrinogen in HCV-infected patients. METHODS: A total of 143 consecutive HCV-infected (RNA⫹) patients (including 57 patients with HCV-related vasculitis) were screened for cryofibrinogen and cryoglobulin (positive if ⬎ 0.05 g/L). The main characteristics and outcome were evaluated according to the cryofibrinogen/cryoglobulin status at baseline. RESULTS: At baseline, 53 of 143 patients (37%) were cryofibrinogen positive, most of whom (47/53 [89%]) were also cryoglobulin positive. Only 37 of 90 cryofibrinogen-negative patients (41%) were cryoglobulin positive (P ⬍ .001). In patients with HCV-related vasculitis, 28 of 57 (49%) were cryofibrinogen positive compared with 25 of 86 patients (29%) without vasculitis (P ⫽ .03). There was a higher rate of renal involvement in cryofibrinogen-negative/cryoglobulin-positive patients than in cryofibrinogen-positive/ cryoglobulin-positive patients (10/25 [40%] vs 3/27 [11%], respectively; P ⫽ .02). After a mean follow-up of 32.6 months, among patients who were cryofibrinogen positive at baseline, 12 of 26 (46%) of those who received an HCV treatment were cryofibrinogen negative at the end of follow-up compared with 4 of 16 (25%) of those who did not receive antiviral drugs. Most patients who became cryofibrinogen negative also became cryoglobulin negative (93%). CONCLUSION: Cryoproteins, including cryoglobulin and cryofibrinogen, are frequently found in the serum of HCV-infected patients. In such patients, a positive cryofibrinogen status is closely related to the presence of cryoglobulin at baseline and after antiviral therapy. © 2008 Elsevier Inc. All rights reserved. • The American Journal of Medicine (2008) 121, 624-631 KEYWORDS: Cryofibrinogen; Cryoglobulin; HCV infection; Vasculitis
Chronic hepatitis C virus (HCV) infection is the leading cause of chronic liver disease in developed countries and may cause cirrhosis and hepatocellular carcinoma. Chronic HCV infection is the main cause of mixed cryoglobulinemia, a systemic vasculitis characterized by the presence of a cryoprotein in the serum, namely, a type II mixed cryoglobulin, and involvement of the skin, kidneys, and nervous system.1-3 Type II mixed cryoglobulinemias are composed
Requests for reprints should be addressed to Patrice Cacoub, MD, AP-HP, Service de Médecine Interne, Hôpital Pitié-Salpêtrière, 83 boulevard de l’hôpital, Paris, F-75013 France. E-mail address: [email protected]
0002-9343/$ -see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.amjmed.2008.03.011
of monoclonal immunoglobulin (Ig)M, which displays antiIgG rheumatoid factor activity, associated with HCV-RNA particles. There is evidence showing that type II mixed cryoglobulinemia is characterized by a monoclonal or oligoclonal proliferation of B cells in bone marrow, liver tissue, and peripheral blood.4-7 HCV infection is also associated with the production of other autoantibodies, such as antinuclear and anticardiolipin antibodies, which reflect chronic nonspecific B-cell proliferation and antigen-driven stimulation.8-13 Cryofibrinogen, another cryoprotein originally characterized in 1955 by Korst and Kratochvil,14 refers to the presence of a cryoprecipitate in plasma that has been anticoag-
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ulated with oxalate, citrate, or edetic acid. This cryoprecipitate 2092 assessments for cryofibrinogen and cryoglobulin were is composed of a complex including fibrin, fibrinogen, fibrin done in 1229 consecutive patients (Figure 1). A total of split products with albumin, fibronectin (cold-insoluble 1035 patients were excluded because of HCV seronegativglobulin), factor VIII, Igs, and other plasma proteins.15 ity. Among the 194 remaining patients, 51 were excluded Cryofibrinogen precipitates in plasma cooled to 4°C and (34 patients who had received HCV treatment before the redissolves on warming to 37°C. cryoprotein assessment, 2 patients Cryofibrinogen is consumed in the who were coinfected with HCV clotting process and therefore and hepatitis B virus or human CLINICAL SIGNIFICANCE does not precipitate in cooled seimmunodeficiency virus, and 15 rum.14 This is in contrast with cryopatients with insufficient data). ● Cryoproteins, including cryoglobulin and globulin, which precipitates in both The mean age, sex ratio, HCV gecryofibrinogen, are frequently found in cooled plasma and serum. Cryofinotype, and rate of vasculitis did the serum of hepatitis C virus-infected brinogenemia may be primary, that not differ between these 51 expatients. is, idiopathic or essential,16-18 or cluded patients and the 143 pa● A positive cryofibrinogen status is closely secondary to an underlying disortients included in the final analysis der.15,16,19-21 Cryofibrinogen may (data not shown). Among the 143 related to the presence of a cryoglobulin induce numerous clinical sympincluded patients, 57 had HCVat baseline and after antiviral therapy. toms, such as cold intolerance, related mixed cryoglobulinemia ● In patients with hepatitis C virus mixed thrombotic and hemorrhagic phevasculitis, defined by the presence cryoglobulinemia, the presence of a cryonomena (eg, purpura, skin necrosis, of typical signs of mixed cryofibrinogen is only associated with less freulcers, and gangrene), and systemic globulinemia vasculitis (eg, asthequent renal involvement, but it has no manifestations (arthralgia and nia, arthralgia, purpura of the impact on vasculitis course. glomerulonephritis),17,22-24 which lower extremities, polyneuropmay mimic mixed cryoglobulineathy, and glomerulonephritis)25 in mia symptoms. the absence of any alternative conThe present study aimed to dedition known to cause vasculitis. termine the prevalence and the clinical, immunologic, and For each patient, clinical and biologic data were recorded therapeutic impacts of cryofibrinogen in HCV-infected paat the time of the initial evaluation (baseline). Clinical tients with or without HCV-related vasculitis. evaluation was based on involvement of the skin (purpura), joints (arthralgia, arthritis), peripheral nervous system (sensory or sensory-motor neuropathy), and kidneys (hematuria, PATIENTS AND METHODS proteinuria, or glomerular filtration rate ⬍ 70 mL/min). LabPatients oratory evaluation included the assessment for and quantification of cryofibrinogen and cryoglobulin. Cryofibrinogen Patients who had a concomitant assessment performed for and cryoglobulin were considered to be positive if greater cryofibrinogen and cryoglobulin were retrospectively identhan 0.05 g/L for both. All patients were positive for HCVtified through the database of the Immunology Laboratory. RNA. Exclusion criteria were the presence of hepatitis B Between January of 1999 and December of 2006, a total of
Figure 1 Study flow chart. *Thirty-four patients treated before the assessment for cryoproteins, 2 coinfected patients with HCV and hepatitis B virus or human immunodeficiency virus, and 15 patients without sufficient data. HCV ⫽ hepatitis C virus.
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surface antigen, anti-human immunodeficiency virus antibodies, or any alternative condition known to cause vasculitis. In 100 of 143 HCV-infected patients with a sufficient follow-up (⬎6 months), the course of the cryoproteins (cryofibrinogen and cryoglobulin), and the virologic and clinical responses to HCV treatment were analyzed. Among them, 54 patients received treatment with interferon alpha ⫾ ribavirin, whereas 46 patients did not receive antiviral drugs. The response to treatment was analyzed by comparing clinical and virologic parameters at the initial evaluation and at the end of follow-up. Clinical response of the vasculitis was defined by analyzing the progression of the following main clinical signs: skin involvement (absence of purpura), peripheral neuropathy (clinical and electrophysiologic improvement on 2 successive examinations), renal involvement (normalization of serum creatinine level and disappearance of proteinuria or hematuria), and absence of arthralgia. A complete clinical response was defined by an improvement in all baseline clinical manifestations. A partial response was defined by an improvement of at least half of the baseline clinical manifestations. All other patients were classified as nonresponders. A sustained virologic response was defined by the absence of detectable serum HCV-RNA 6 months after stopping antiviral treatment; the remaining patients were classified as nonsustained virologic responders. HCV antibodies were detected by 2 specific third-generation immunoassays (Monolisa anti-HCV Plus, Sanofi Diagnostic Pasteur, Marne la Coquette, France; Axsym HCV version 3.0, Abbott, Les Ulys, France). Serum HCV-RNA was measured by a reverse-transcription polymerase chain reaction assay (Amplicor HCV test, Roche Diagnostics, Neuilly/Seine, France). HCV genotyping was performed using a second-generation line probe assay (LiPA; Innogenetics, Brussels, Belgium). Liver biopsy specimens, if done, were evaluated according to the previously validated Metavir scoring system.26 We determined the plasma fibrinogen level by the nephelometric chromometric method (BNA, Dade-Behring, Marburg, Germany).27
read in a spectrophotometer at 280 nm. Quantification of cryoglobulin was calculated according to a standard curve (obtained using a purified human gamma-globulin preparation supplied by the Etablissement Fançais du Sang, Paris, France). Similarly, a purified fibrinogen preparation was used for cryofibrinogen quantification. Characterization of purified cryoprecipitate components was performed by western blotting after thin-layer agarose electrophoresis was performed as previously described.28 Briefly, cryoglobulin and cryofibrinogen, at a concentration of 0.05 mg/mL in 0.15 mol/L NaCl (pH 7.4), were separated by electrophoresis at 37°C on thin-layer agarose gels (Paragon, Beckman, Gagny, France) and transferred onto nitrocellulose sheets (TransBlot Transfer medium, BioRad Laboratories, Calif) by pressure blotting (pressure of 1 kg and then 5 kg, each for 5 minutes). The blots were then ovendried. After saturation with 50 mg/mL skimmed milk (Régilait, Lyon, France) in 0.15 mol/L NaCl for 1 hour at 37°C, the blots were probed with polyclonal antibodies (rabbit antibodies, diluted 1/3000) (Dako, Trappes, France) specific for either fibrinogen or light and heavy chains of human Ig. After four 5-minute washes in 0.15 mol/L NaCl, the strips were incubated for 30 minutes with a 5000-fold dilution of the appropriate detection antibody, that is, goat anti-rabbit antibody labeled with alkaline phosphatase (EC 3.1.3.1, Jackson ImmunoResearch Labs Inc, West Grove, Pa). After further washes, enzyme activity was revealed using the appropriate substrate (BCIP/NBT: Sigma, St Louis, Mo) prepared just before use. Concentrations as low as 20 g/mL of a monoclonal Ig of the various isotypes (IgG, IgA, and IgM) were easily detected, as was fibrinogen.
Analysis of Cryoproteins
The main characteristics of the 143 patients included in the analysis are detailed in Table 1. Their mean age was 56.6 ⫾ 14.3 years (range 14.3-83.1 years), and 70 patients (49%) were male. The most frequent HCV genotype was genotype 1 (n ⫽ 66 [46%]). Mean serum fibrinogen value was 3.29 ⫾ 0.92 g/L (range 1.4-6.3 g/L).
With the use of prewarmed equipment, 10 mL of blood was collected into either anticoagulant-free tubes for cryoglobulin detection or citrated tubes for cryofibrinogen detection. Both sera (for cryoglobulin screening) and plasma (for cryofibrinogen screening) were prepared by centrifuging at 3000g for 30 minutes at 37°C. An antiseptic (sodium azide, 0.1 mg/L) was then added. The sera/plasma were chilled to 4°C for 7 days and analyzed for precipitate or gel formation. By warming the precipitation to 37°C, we checked the reversible nature of the cryofibrinogen precipitate before proceeding with a new precipitation at 4°C and purification. Cryoglobulin and cryofibrinogen were isolated by centrifugation (3000g for 15 minutes) and then extensively washed in a minimal volume of 0.15 mol/L NaCl (pH 7.4). A fraction of the washed cryoglobulin and cryofibrinogen was redissolved in 0.1 mol/L NaOH, and the absorbance was
Statistical Analysis Quantitative data are shown as means ⫾ standard deviation. The Fisher exact test was used to compare qualitative variables. Spearman correlation rank was used to test the correlation between quantitative variables.
RESULTS
Cryofibrinogen and Cryoglobulin Status at Baseline At baseline, 53 of 143 patients (37%) were cryofibrinogen positive. There were more cryofibrinogen-positive/cryoglobulin-positive patients (47/53 [89%]) than cryofibrinogen-negative/cryoglobulin-positive patients (37/90 [41%]) (P ⬍ .001) (Table 2). Only 6 of 143 patients (4%) had an isolated cryofibrinogen. The rate of cryofibrinogen positivity was higher in patients with HCV-related vasculitis (28/57 [49%]) than in HCV-infected patients without vas-
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Table 1 Main Characteristics of Hepatitis C Virus-Infected Patients According to the Presence of a Vasculitis and the Type of Cryoprotein Parameter
Total N ⫽ 143
HCV-Related Vasculitis N ⫽ 57
HCV-Infected Patients Without Vasculitis N ⫽ 86
Age, mean ⫾ SD (y) Male gender, n (%) HCV ALT level, ULN Genotype 1 Liver necroinflammation (0-3)* Liver fibrosis (0-4)* Cryoprotein Cryoglobulinemia of type II Level (mean ⫾ SD, g/L) Cryoglobulinemia of type III Level (mean ⫾ SD, g/L) Cryofibrinogenemia Level (mean ⫾ SD, g/L)
56.6 ⫾ 14.3 70 (49%)
60.4 ⫾ 13.4 32 (56%)
54.1 ⫾ 14.4 38 (44%)
.005 .18
2.18 ⫾ 1.61 66 (46%) 1.24 ⫾ 0.84 1.67 ⫾ 1.27
2.21 ⫾ 1.63 35 (61%) 1.42 ⫾ 0.94 1.98 ⫾ 1.29
2.16 ⫾ 1.61 31 (36%) 1.10 ⫾ 0.73 1.45 ⫾ 1.21
.43 .004 .02 .01
52 (36%) 0.83 ⫾ 1.03 28 (20%) 0.35 ⫾ 0.50 53 (37%) 0.52 ⫾ 0.77
39 (68%) 0.97 ⫾ 1.11 9 (16%) 0.54 ⫾ 0.80 28 (49%) 0.78 ⫾ 0.98
15 (16%) 0.40 ⫾ 0.58 17 (20%) 0.25 ⫾ 0.19 25 (29%) 0.23 ⫾ 0.17
⬍.001 .04 .66 .08 .02 .006
P
HCV ⫽ hepatitis C virus; ALT ⫽ alanine aminotransferase; ULN ⫽ upper limits of normal; SD ⫽ standard deviation. *Grading of liver necroinflammation (A) and fibrosis (F) according to the Metavir score.26
culitis (25/86 [29%]) (P ⬍ .03). There was a significant correlation between cryoglobulin and cryofibrinogen levels, as shown in Figure 2 (r2 ⫽ 0.764, P ⬍ .001 for the entire cohort of HCV-infected patients; and r2 ⫽ 0.759, P ⬍ .001 for patients with HCV-related vasculitis). There was no correlation between cryofibrinogen levels and fibrinogen levels (data not shown).
Course of Cryofibrinogen and Cryoglobulin During the Follow-Up Among the 100 patients who had a follow-up more than 6 months (mean 32.6 ⫾ 21.5, 6-85 months), 54 received an HCV treatment: peg-interferon alpha-2b (1.5 g/kg/wk) plus ribavirin (600-1200 mg/d) (n ⫽ 43); interferon alpha-2b (3 million IU 3 times/wk) plus ribavirin (600-1200 mg/s) (n ⫽ 8); ribavirin alone (600-1200 mg/d) (n ⫽ 2); or interferon alpha-2b alone (3 million IU 3 times/wk) (n ⫽ 1). The course of cryofibrinogen status in treated patients, between baseline and the end of follow-up, is detailed in Figure 3. Among patients who showed a sustained virologic response, 9 of 19 cryofibrinogen-positive patients (47%) at baseline became cryofibrinogen negative at the end of follow-up, whereas 10 of 13 cryofibrinogen-negative patients
Table 2
(77%) at baseline remained cryofibrinogen negative at the end of follow-up (P ⫽ .15). All of these cryofibrinogenpositive patients at baseline also were cryoglobulin positive. In nonsustained virologic responders patients, 3 of 7 cryofibrinogen-positive patients (43%) at baseline became cryofibrinogen negative at the end of follow-up, whereas 10 of 15 cryofibrinogen-negative patients (67%) at baseline remained cryofibrinogen negative at the end of follow-up (P ⫽ .38). For patients who did not receive HCV treatment (n ⫽ 44), 4 of 16 cryofibrinogen-positive patients (25%) at baseline became cryofibrinogen negative at the end of follow-up, whereas 19 of 28 cryofibrinogen-negative patients (68%) at baseline remained cryofibrinogen negative at the end of follow-up (P ⫽ .01). Among cryofibrinogen-positive patients at baseline, 12 of 26 (46%) of those who received an HCV treatment became cryofibrinogen negative at the end of follow-up compared with 4 of 16 (25%) of those who did not receive antiviral drugs (P ⫽ .2). Respective rates for cryofibrinogen-negative patients at baseline were 22 of 30 (73%) and 19 of 28 (68%) (P ⫽ .7). In the entire cohort of HCV-infected patients, most cryofibrinogen-positive patients at baseline also were cryoglobulin positive (39/42 [93%]), and those who became cryofi-
Cryofibrinogen and Cryoglobulin Status in Hepatitis C Virus-Infected Patients at Baseline
CF positive, n (%) CF negative, n (%)
All Patients (N ⫽ 143)
HCV-Related Vasculitis (n ⫽ 57)
HCV-Infected Patients Without Vasculitis (n ⫽ 86)
CG⫹
CG⫺
CG⫹
CG⫺
CG⫹
CG⫺
47 (89%) 37 (41%)
6 (11%) 53 (59%)
27 (96%) 25 (86%)
1 (4%) 4 (14%)
20 (80%) 12 (20%)
5 (20%) 49 (80%)
HCV ⫽ hepatitis C virus; CF ⫽ cryofibrinogen.
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Figure 2 Spearman rank correlation between cryofibrinogen and cryoglobulin serum levels in the entire cohort of 143 HCV-infected patients (r2 ⫽ 0.764, P ⬍ .001) (left) and in 57 patients with HCV-related vasculitis (r2 ⫽ 0.759, P ⬍ .001) (right). CF ⫽ cryofibrinogen; CG ⫽ cryoglobulin.
Figure 3 Course of Cryofibrinogen Status in Patients Who Received Hepatitis C Virus Treatment.
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Table 3 Baseline Manifestations and Outcomes of Patients with Hepatitis C Virus-Related Vasculitis Who Received Hepatitis C Virus Treatment, According to the Cryofibrinogen/Cryoglobulin Status at Baseline P Cryoprotein Status at Baseline Cryofibrinogen Cryoglobulin Manifestations of the Vasculitis at Baseline Skin, n (%) Nerve Kidney Joints Response to Hepatitis C Virus Treatment Clinical response of the vasculitis Complete response Partial response Nonresponse Sustained virologic response
⫹ ⫹ 27/57 21/27 19/27 3/27 12/27
15/23 7/23 1/23 16/23
(47%)* (78%) (70%) (11%) (44%)
⫺ ⫹ 25/57 19/25 16/25 10/25 13/25
(44%)* (76%) (64%) (40%) (52%)
1.00 .77 .02 .78
(65%) (30%) (4%) (70%)
11/19 3/19 4/19 11/19
(58%) (16%) (21%) (58%)
.75 .30 .16 .52
*Among the patients with hepatitis C virus-related vasculitis, 4 were cryofibrinogen negative/cryoglobulin negative and 1 was cryofibrinogen positive/cryoglobulin negative.
brinogen negative at the end of follow-up also became cryoglobulin negative (13/14 [93%]), whereas 24 of 25 patients (96%) who remained cryofibrinogen positive also remained cryoglobulin positive. Among treated patients, 10 of 11 patients (91%) who became cryofibrinogen negative also became cryoglobulin negative, whereas 13 of 14 (93%) of those who remained cryofibrinogen positive also remained cryoglobulin positive.
Analysis of Patients with HCV-Related Vasculitis Fifty-seven patients had HCV-related vasculitis. The main clinical manifestations at baseline and the clinical and virologic outcomes according to cryoprotein status at baseline are detailed in Table 3. Clinical manifestations of HCVrelated vasculitis did not differ in the 27 cryofibrinogenpositive/cryoglobulin-positive patients compared with the 25 cryofibrinogen-negative/cryoglobulin-positive patients, except for a lower rate of renal involvement (40% vs 11%, respectively; P ⫽ .02). The rate of non-Hodgkin’s lymphoma was 8% (2/25) in cryofibrinogen-positive/cryoglobulin-positive patients versus 15% (4/27) in cryofibrinogen-negative/ cryoglobulin-positive patients (P ⫽ not significant). Five patients had clinical signs of HCV-related vasculitis without identification of cryoglobulin at baseline. Four patients were cryofibrinogen negative/cryoglobulin negative, and their vasculitis involved the skin (1), nerves (3), and kidneys (1). One patient who was cryofibrinogen positive/ cryoglobulin negative at baseline presented with peripheral nerve involvement. Forty-eight patients (84%) with vasculitis had a sufficient follow-up (mean 32.9 ⫾ 23.0 months [6-87]) for the analysis of clinical, immunologic, and virologic responses. There were no significant differences in clinical and virologic
responses between cryofibrinogen-positive/cryoglobulinpositive and cryofibrinogen-negative/cryoglobulin-positive patients. Among the cryofibrinogen-positive/cryoglobulinpositive patients at baseline, 10 became cryofibrinogen negative at the end of follow-up and also became cryoglobulin negative. Among the 14 patients who remained cryofibrinogen positive at the end of follow-up, only 1 patient became cryoglobulin negative. Fourteen patients had a renal involvement, with a biopsyproven membranoproliferative glomerulonephritis in 10 of 14 patients. In the 4 other patients, renal biopsy was not performed because a peripheral nerve biopsy already showed signs of cryoglobulin vasculitis. Among these patients, 3 of 14 were cryoglobulin positive/cryofibrinogen positive at baseline; 2 of 3 patients had a partial response and 1 of 3 patients had a complete response of the vasculitis after treatment. One patient was cryoglobulin negative/ cryofibrinogen negative at baseline and had a complete response of the vasculitis after treatment.
DISCUSSION Most HCV-infected patients (40%-60%) produce mixed cryoglobulin, a circulating serum cryoprotein composed of Igs and HCV-RNA particles. However, only a minority of HCV mixed cryoglobulin-positive patients will develop systemic vasculitis involving the skin, kidneys, and nervous system. The underlying mechanisms are not completely understood. Cryofibrinogen is another cryoprotein complex including fibrin, fibrinogen, factor VIII, and sometimes Ig. Cryofibrinogen can mimic some cryoglobulin manifestations, such as cold intolerance, purpura, skin necrosis, ulcers, and gangrene.17,22-24 Our study was designed to determine whether the presence of a cryofibrinogen may worsen mixed cryoglobulinemia-related vasculitis. In the
630 present study, we found that cryoproteins, including cryoglobulin and cryofibrinogen, are frequently found in the serum of HCV-infected patients. Cryofibrinogen was almost always found when cryoglobulin was present. Cryofibrinogen was more frequently found in patients with HCVrelated vasculitis than in those without vasculitis (49% vs 29%). In these patients, the presence of a cryofibrinogen was associated with a lower rate of renal involvement. Although antiviral therapy decreased the rate of cryofibrinogen positivity, the presence of a cryofibrinogen had no impact on clinical and virologic responses to antiviral therapy. The presence of a cryofibrinogen has been suggested as a marker of the severity of the underlying disease. A study of 284 patients with inflammatory bowel disease found that elevated cryofibrinogen was associated with a more severe disease and refractoriness to medication.29 Another study showed a 30% increased level of cryofibrinogen in 53 cases of nephrosis (24 cases) and acute and chronic renal failure (17 and 12 patients, respectively).30 A cryofibrinogen may cause intravascular coagulation in which fibrin can be deposited in the glomerular filters and may cause anuria, and in the renal vasculature where it may cause ischemic tubular necrosis. Other studies,22,28,31 however, have shown no correlation between levels of cryofibrinogen and disease intensity. In the present study, clinical signs and response to treatment were similar, regardless of the cryofibrinogen status at baseline. The only significant difference was that patients with vasculitis without cryofibrinogen had a higher rate of renal involvement. However, because there were few patients with renal involvement, definite conclusions cannot be drawn from our study. As observed with cryoglobulin,32,33 antiviral therapy decreased the incidence of cryofibrinogen positivity. This might be the result of the disappearance of cryoglobulin. The presence of a cryofibrinogen was strongly correlated with a positive cryoglobulin. Some 93% of cryofibrinogen-positive patients also were cryoglobulin positive, and cryofibrinogen levels were positively correlated with cryoglobulin levels. The same observation was made during follow-up. At the end of the study, a large majority of patients who became cryofibrinogen negative also were cryoglobulin negative. Taken together, these data support the hypothesis that cryofibrinogen may be linked to the presence of a cryoglobulin. The absence of a clinical and virologic impact of a cryofibrinogen in patients with vasculitis suggests that the presence of a cryofibrinogen may be a phenomenon linked to the presence of a cryoglobulin. We may ask if a cryofibrinogen is really a separate protein or a form of cryoglobulin that binds to fibrins products in the serum. Many arguments support that these are really 2 different cryoproteins. Only 56% of cryoglobulin-positive patients at baseline (and 52% of patients with HCV-related vasculitis) also were cryofibrinogen positive. Cryofibrinogen and cryoglobulin are clearly identified as 2 different cryoproteins with our
The American Journal of Medicine, Vol 121, No 7, July 2008 laboratory tests. A cryofibrinogen can be found without cryoglobulin with a particular clinical course.28
CONCLUSIONS Cryoproteins, including cryoglobulin and cryofibrinogen, are frequently found in the serum of HCV-infected patients. A cryofibrinogen is almost always found when a cryoglobulin is present. The presence of a cryofibrinogen has no impact on clinical and virologic responses to antiviral therapy. In such patients, positive cryofibrinogen status is closely related to the presence of a cryoglobulin at baseline and after antiviral therapy. Further studies are needed to determine whether the presence of a cryofibrinogen is associated with a lower rate of renal involvement in patients with mixed cryoglobulinemia-related vasculitis.
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16. Rachmilewitz EA, Sacks MI, Zlotnick A. Essential cryofibrinogenemia. Clinical, pathological and immunological studies. Isr J Med Sci. 1970;6:32-43. 17. Beightler E, Diven DG, Sanchez RL, Solomon AR. Thrombotic vasculopathy associated with cryofibrinogenemia. J Am Acad Dermatol. 1991;24(2 Pt 2):342-345. 18. Jantunen E, Soppi E, Neittaanmaki H, Lahtinen R. Essential cryofibrinogenaemia, leukocytoclastic vasculitis and chronic purpura. J Intern Med. 1993;234:331-333. 19. Zlotnick A, Shahin W, Rachmilewitz EA. Studies in cryofibrinogenemia. Acta Haematol. 1969;42:8-17. 20. Smith SB, Arkin C. Cryofibrinogenemia: incidence, clinical correlations, and a review of the literature. Am J Clin Pathol. 1972;58:524-530. 21. Robinson MG, Troiano G, Cohen H, Foadi M. Acute transient cryofibrinogenemia in infants. J Pediatr. 1966;69:35-39. 22. Ritzmann SE, Levin WC, Ivers JB, Koch JL. I. Cryoproteinemias. 2. Primary cryofibrinogenemia—its association with cryopathy and telangiectasis. Tex Rep Biol Med. 1963;21:567-580. 23. Ireland TA, Werner DA, Rietschel RL, et al. Cutaneous lesions in cryofibrinogenemia. J Pediatr. 1984;105:67-69. 24. Nagy J, Ambrus M, Paal M, et al. Cryoglobulinaemia and cryofibrinogenaemia in IgA nephropathy: a follow-up study. Nephron. 1987;46: 337-342.
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25. Meltzer M, Franklin EC, Elias K, et al. Cryoglobulinemia—a clinical and laboratory study. II. Cryoglobulins with rheumatoid factor activity. Am J Med. 1966;40:837-856. 26. The French METAVIR Cooperative Study Group. Intraobserver and interobserver variations in liver biopsy interpretation in patients with chronic hepatitis C. Hepatology. 1994;20(1 Pt 1):15-20. 27. Von Clauss A. Gerinnungsphysiologische Scnellmethod zur bestimmung des fibrinogens. Acta Haematol. 1957;17:237-246. 28. Blain H, Cacoub P, Musset L, et al. Cryofibrinogenaemia: a study of 49 patients. Clin Exp Immunol. 2000;120:253-260. 29. Sawada K, Takahashi R, Saniabadi, et al. Elevated plasma cryofibrinogen in patients with active inflammatory bowel disease is morbigenous. World J Gastroenterol. 2006;12:1621-1625. 30. Wardle EN, Taylor G. Fibrin breakdown products and fibrinolysis in renal disease. J Clin Pathol. 1968;21:140-146. 31. Kuipers JG, Kellett J, May D. Low levels of cryofibrinogenaemia and peripheral circulatory dysfunction. Ir Med J. 1991;84:68-69. 32. Cacoub P, Ratziu V, Myers RP, et al. Impact of treatment on extra hepatic manifestations in patients with chronic hepatitis C. J Hepatol. 2002;36:812-818. 33. Saadoun D, Resche-Rigon M, Thibault V, et al. Antiviral therapy for hepatitis C virus-associated mixed cryoglobulinemia vasculitis: a longterm followup study. Arthritis Rheum. 2006;54:3696-3706.
Cryofibrinogen in Patients with Hepatitis C Virus Infection Aurélien Delluc, MD,a David Saadoun, MD, PhD,a Pascale Ghillani-Dalbin, PhD,b Damien Sene, MD, PhD,a Jean Charles Piette, MD,a Patrice Cacoub, MD, PhDa a
Service de Médecine Interne, Université Pierre et Marie Curie, Paris 6, France; bLaboratoire d’Immunochimie, Hôpital Pitié-Salpêtrière, Paris, France.
ABSTRACT BACKGROUND: Mixed cryoglobulin is usually associated with hepatitis C virus (HCV) infection and might cause systemic vasculitis. The presence and impact of cryofibrinogen, another cryoprotein, in the serum of HCV-infected patients have not yet been evaluated. The objective was to study the prevalence and the clinical and therapeutic impacts of cryofibrinogen in HCV-infected patients. METHODS: A total of 143 consecutive HCV-infected (RNA⫹) patients (including 57 patients with HCV-related vasculitis) were screened for cryofibrinogen and cryoglobulin (positive if ⬎ 0.05 g/L). The main characteristics and outcome were evaluated according to the cryofibrinogen/cryoglobulin status at baseline. RESULTS: At baseline, 53 of 143 patients (37%) were cryofibrinogen positive, most of whom (47/53 [89%]) were also cryoglobulin positive. Only 37 of 90 cryofibrinogen-negative patients (41%) were cryoglobulin positive (P ⬍ .001). In patients with HCV-related vasculitis, 28 of 57 (49%) were cryofibrinogen positive compared with 25 of 86 patients (29%) without vasculitis (P ⫽ .03). There was a higher rate of renal involvement in cryofibrinogen-negative/cryoglobulin-positive patients than in cryofibrinogen-positive/ cryoglobulin-positive patients (10/25 [40%] vs 3/27 [11%], respectively; P ⫽ .02). After a mean follow-up of 32.6 months, among patients who were cryofibrinogen positive at baseline, 12 of 26 (46%) of those who received an HCV treatment were cryofibrinogen negative at the end of follow-up compared with 4 of 16 (25%) of those who did not receive antiviral drugs. Most patients who became cryofibrinogen negative also became cryoglobulin negative (93%). CONCLUSION: Cryoproteins, including cryoglobulin and cryofibrinogen, are frequently found in the serum of HCV-infected patients. In such patients, a positive cryofibrinogen status is closely related to the presence of cryoglobulin at baseline and after antiviral therapy. © 2008 Elsevier Inc. All rights reserved. • The American Journal of Medicine (2008) 121, 624-631 KEYWORDS: Cryofibrinogen; Cryoglobulin; HCV infection; Vasculitis
Chronic hepatitis C virus (HCV) infection is the leading cause of chronic liver disease in developed countries and may cause cirrhosis and hepatocellular carcinoma. Chronic HCV infection is the main cause of mixed cryoglobulinemia, a systemic vasculitis characterized by the presence of a cryoprotein in the serum, namely, a type II mixed cryoglobulin, and involvement of the skin, kidneys, and nervous system.1-3 Type II mixed cryoglobulinemias are composed
Requests for reprints should be addressed to Patrice Cacoub, MD, AP-HP, Service de Médecine Interne, Hôpital Pitié-Salpêtrière, 83 boulevard de l’hôpital, Paris, F-75013 France. E-mail address: [email protected]
0002-9343/$ -see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.amjmed.2008.03.011
of monoclonal immunoglobulin (Ig)M, which displays antiIgG rheumatoid factor activity, associated with HCV-RNA particles. There is evidence showing that type II mixed cryoglobulinemia is characterized by a monoclonal or oligoclonal proliferation of B cells in bone marrow, liver tissue, and peripheral blood.4-7 HCV infection is also associated with the production of other autoantibodies, such as antinuclear and anticardiolipin antibodies, which reflect chronic nonspecific B-cell proliferation and antigen-driven stimulation.8-13 Cryofibrinogen, another cryoprotein originally characterized in 1955 by Korst and Kratochvil,14 refers to the presence of a cryoprecipitate in plasma that has been anticoag-
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ulated with oxalate, citrate, or edetic acid. This cryoprecipitate 2092 assessments for cryofibrinogen and cryoglobulin were is composed of a complex including fibrin, fibrinogen, fibrin done in 1229 consecutive patients (Figure 1). A total of split products with albumin, fibronectin (cold-insoluble 1035 patients were excluded because of HCV seronegativglobulin), factor VIII, Igs, and other plasma proteins.15 ity. Among the 194 remaining patients, 51 were excluded Cryofibrinogen precipitates in plasma cooled to 4°C and (34 patients who had received HCV treatment before the redissolves on warming to 37°C. cryoprotein assessment, 2 patients Cryofibrinogen is consumed in the who were coinfected with HCV clotting process and therefore and hepatitis B virus or human CLINICAL SIGNIFICANCE does not precipitate in cooled seimmunodeficiency virus, and 15 rum.14 This is in contrast with cryopatients with insufficient data). ● Cryoproteins, including cryoglobulin and globulin, which precipitates in both The mean age, sex ratio, HCV gecryofibrinogen, are frequently found in cooled plasma and serum. Cryofinotype, and rate of vasculitis did the serum of hepatitis C virus-infected brinogenemia may be primary, that not differ between these 51 expatients. is, idiopathic or essential,16-18 or cluded patients and the 143 pa● A positive cryofibrinogen status is closely secondary to an underlying disortients included in the final analysis der.15,16,19-21 Cryofibrinogen may (data not shown). Among the 143 related to the presence of a cryoglobulin induce numerous clinical sympincluded patients, 57 had HCVat baseline and after antiviral therapy. toms, such as cold intolerance, related mixed cryoglobulinemia ● In patients with hepatitis C virus mixed thrombotic and hemorrhagic phevasculitis, defined by the presence cryoglobulinemia, the presence of a cryonomena (eg, purpura, skin necrosis, of typical signs of mixed cryofibrinogen is only associated with less freulcers, and gangrene), and systemic globulinemia vasculitis (eg, asthequent renal involvement, but it has no manifestations (arthralgia and nia, arthralgia, purpura of the impact on vasculitis course. glomerulonephritis),17,22-24 which lower extremities, polyneuropmay mimic mixed cryoglobulineathy, and glomerulonephritis)25 in mia symptoms. the absence of any alternative conThe present study aimed to dedition known to cause vasculitis. termine the prevalence and the clinical, immunologic, and For each patient, clinical and biologic data were recorded therapeutic impacts of cryofibrinogen in HCV-infected paat the time of the initial evaluation (baseline). Clinical tients with or without HCV-related vasculitis. evaluation was based on involvement of the skin (purpura), joints (arthralgia, arthritis), peripheral nervous system (sensory or sensory-motor neuropathy), and kidneys (hematuria, PATIENTS AND METHODS proteinuria, or glomerular filtration rate ⬍ 70 mL/min). LabPatients oratory evaluation included the assessment for and quantification of cryofibrinogen and cryoglobulin. Cryofibrinogen Patients who had a concomitant assessment performed for and cryoglobulin were considered to be positive if greater cryofibrinogen and cryoglobulin were retrospectively identhan 0.05 g/L for both. All patients were positive for HCVtified through the database of the Immunology Laboratory. RNA. Exclusion criteria were the presence of hepatitis B Between January of 1999 and December of 2006, a total of
Figure 1 Study flow chart. *Thirty-four patients treated before the assessment for cryoproteins, 2 coinfected patients with HCV and hepatitis B virus or human immunodeficiency virus, and 15 patients without sufficient data. HCV ⫽ hepatitis C virus.
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surface antigen, anti-human immunodeficiency virus antibodies, or any alternative condition known to cause vasculitis. In 100 of 143 HCV-infected patients with a sufficient follow-up (⬎6 months), the course of the cryoproteins (cryofibrinogen and cryoglobulin), and the virologic and clinical responses to HCV treatment were analyzed. Among them, 54 patients received treatment with interferon alpha ⫾ ribavirin, whereas 46 patients did not receive antiviral drugs. The response to treatment was analyzed by comparing clinical and virologic parameters at the initial evaluation and at the end of follow-up. Clinical response of the vasculitis was defined by analyzing the progression of the following main clinical signs: skin involvement (absence of purpura), peripheral neuropathy (clinical and electrophysiologic improvement on 2 successive examinations), renal involvement (normalization of serum creatinine level and disappearance of proteinuria or hematuria), and absence of arthralgia. A complete clinical response was defined by an improvement in all baseline clinical manifestations. A partial response was defined by an improvement of at least half of the baseline clinical manifestations. All other patients were classified as nonresponders. A sustained virologic response was defined by the absence of detectable serum HCV-RNA 6 months after stopping antiviral treatment; the remaining patients were classified as nonsustained virologic responders. HCV antibodies were detected by 2 specific third-generation immunoassays (Monolisa anti-HCV Plus, Sanofi Diagnostic Pasteur, Marne la Coquette, France; Axsym HCV version 3.0, Abbott, Les Ulys, France). Serum HCV-RNA was measured by a reverse-transcription polymerase chain reaction assay (Amplicor HCV test, Roche Diagnostics, Neuilly/Seine, France). HCV genotyping was performed using a second-generation line probe assay (LiPA; Innogenetics, Brussels, Belgium). Liver biopsy specimens, if done, were evaluated according to the previously validated Metavir scoring system.26 We determined the plasma fibrinogen level by the nephelometric chromometric method (BNA, Dade-Behring, Marburg, Germany).27
read in a spectrophotometer at 280 nm. Quantification of cryoglobulin was calculated according to a standard curve (obtained using a purified human gamma-globulin preparation supplied by the Etablissement Fançais du Sang, Paris, France). Similarly, a purified fibrinogen preparation was used for cryofibrinogen quantification. Characterization of purified cryoprecipitate components was performed by western blotting after thin-layer agarose electrophoresis was performed as previously described.28 Briefly, cryoglobulin and cryofibrinogen, at a concentration of 0.05 mg/mL in 0.15 mol/L NaCl (pH 7.4), were separated by electrophoresis at 37°C on thin-layer agarose gels (Paragon, Beckman, Gagny, France) and transferred onto nitrocellulose sheets (TransBlot Transfer medium, BioRad Laboratories, Calif) by pressure blotting (pressure of 1 kg and then 5 kg, each for 5 minutes). The blots were then ovendried. After saturation with 50 mg/mL skimmed milk (Régilait, Lyon, France) in 0.15 mol/L NaCl for 1 hour at 37°C, the blots were probed with polyclonal antibodies (rabbit antibodies, diluted 1/3000) (Dako, Trappes, France) specific for either fibrinogen or light and heavy chains of human Ig. After four 5-minute washes in 0.15 mol/L NaCl, the strips were incubated for 30 minutes with a 5000-fold dilution of the appropriate detection antibody, that is, goat anti-rabbit antibody labeled with alkaline phosphatase (EC 3.1.3.1, Jackson ImmunoResearch Labs Inc, West Grove, Pa). After further washes, enzyme activity was revealed using the appropriate substrate (BCIP/NBT: Sigma, St Louis, Mo) prepared just before use. Concentrations as low as 20 g/mL of a monoclonal Ig of the various isotypes (IgG, IgA, and IgM) were easily detected, as was fibrinogen.
Analysis of Cryoproteins
The main characteristics of the 143 patients included in the analysis are detailed in Table 1. Their mean age was 56.6 ⫾ 14.3 years (range 14.3-83.1 years), and 70 patients (49%) were male. The most frequent HCV genotype was genotype 1 (n ⫽ 66 [46%]). Mean serum fibrinogen value was 3.29 ⫾ 0.92 g/L (range 1.4-6.3 g/L).
With the use of prewarmed equipment, 10 mL of blood was collected into either anticoagulant-free tubes for cryoglobulin detection or citrated tubes for cryofibrinogen detection. Both sera (for cryoglobulin screening) and plasma (for cryofibrinogen screening) were prepared by centrifuging at 3000g for 30 minutes at 37°C. An antiseptic (sodium azide, 0.1 mg/L) was then added. The sera/plasma were chilled to 4°C for 7 days and analyzed for precipitate or gel formation. By warming the precipitation to 37°C, we checked the reversible nature of the cryofibrinogen precipitate before proceeding with a new precipitation at 4°C and purification. Cryoglobulin and cryofibrinogen were isolated by centrifugation (3000g for 15 minutes) and then extensively washed in a minimal volume of 0.15 mol/L NaCl (pH 7.4). A fraction of the washed cryoglobulin and cryofibrinogen was redissolved in 0.1 mol/L NaOH, and the absorbance was
Statistical Analysis Quantitative data are shown as means ⫾ standard deviation. The Fisher exact test was used to compare qualitative variables. Spearman correlation rank was used to test the correlation between quantitative variables.
RESULTS
Cryofibrinogen and Cryoglobulin Status at Baseline At baseline, 53 of 143 patients (37%) were cryofibrinogen positive. There were more cryofibrinogen-positive/cryoglobulin-positive patients (47/53 [89%]) than cryofibrinogen-negative/cryoglobulin-positive patients (37/90 [41%]) (P ⬍ .001) (Table 2). Only 6 of 143 patients (4%) had an isolated cryofibrinogen. The rate of cryofibrinogen positivity was higher in patients with HCV-related vasculitis (28/57 [49%]) than in HCV-infected patients without vas-
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Table 1 Main Characteristics of Hepatitis C Virus-Infected Patients According to the Presence of a Vasculitis and the Type of Cryoprotein Parameter
Total N ⫽ 143
HCV-Related Vasculitis N ⫽ 57
HCV-Infected Patients Without Vasculitis N ⫽ 86
Age, mean ⫾ SD (y) Male gender, n (%) HCV ALT level, ULN Genotype 1 Liver necroinflammation (0-3)* Liver fibrosis (0-4)* Cryoprotein Cryoglobulinemia of type II Level (mean ⫾ SD, g/L) Cryoglobulinemia of type III Level (mean ⫾ SD, g/L) Cryofibrinogenemia Level (mean ⫾ SD, g/L)
56.6 ⫾ 14.3 70 (49%)
60.4 ⫾ 13.4 32 (56%)
54.1 ⫾ 14.4 38 (44%)
.005 .18
2.18 ⫾ 1.61 66 (46%) 1.24 ⫾ 0.84 1.67 ⫾ 1.27
2.21 ⫾ 1.63 35 (61%) 1.42 ⫾ 0.94 1.98 ⫾ 1.29
2.16 ⫾ 1.61 31 (36%) 1.10 ⫾ 0.73 1.45 ⫾ 1.21
.43 .004 .02 .01
52 (36%) 0.83 ⫾ 1.03 28 (20%) 0.35 ⫾ 0.50 53 (37%) 0.52 ⫾ 0.77
39 (68%) 0.97 ⫾ 1.11 9 (16%) 0.54 ⫾ 0.80 28 (49%) 0.78 ⫾ 0.98
15 (16%) 0.40 ⫾ 0.58 17 (20%) 0.25 ⫾ 0.19 25 (29%) 0.23 ⫾ 0.17
⬍.001 .04 .66 .08 .02 .006
P
HCV ⫽ hepatitis C virus; ALT ⫽ alanine aminotransferase; ULN ⫽ upper limits of normal; SD ⫽ standard deviation. *Grading of liver necroinflammation (A) and fibrosis (F) according to the Metavir score.26
culitis (25/86 [29%]) (P ⬍ .03). There was a significant correlation between cryoglobulin and cryofibrinogen levels, as shown in Figure 2 (r2 ⫽ 0.764, P ⬍ .001 for the entire cohort of HCV-infected patients; and r2 ⫽ 0.759, P ⬍ .001 for patients with HCV-related vasculitis). There was no correlation between cryofibrinogen levels and fibrinogen levels (data not shown).
Course of Cryofibrinogen and Cryoglobulin During the Follow-Up Among the 100 patients who had a follow-up more than 6 months (mean 32.6 ⫾ 21.5, 6-85 months), 54 received an HCV treatment: peg-interferon alpha-2b (1.5 g/kg/wk) plus ribavirin (600-1200 mg/d) (n ⫽ 43); interferon alpha-2b (3 million IU 3 times/wk) plus ribavirin (600-1200 mg/s) (n ⫽ 8); ribavirin alone (600-1200 mg/d) (n ⫽ 2); or interferon alpha-2b alone (3 million IU 3 times/wk) (n ⫽ 1). The course of cryofibrinogen status in treated patients, between baseline and the end of follow-up, is detailed in Figure 3. Among patients who showed a sustained virologic response, 9 of 19 cryofibrinogen-positive patients (47%) at baseline became cryofibrinogen negative at the end of follow-up, whereas 10 of 13 cryofibrinogen-negative patients
Table 2
(77%) at baseline remained cryofibrinogen negative at the end of follow-up (P ⫽ .15). All of these cryofibrinogenpositive patients at baseline also were cryoglobulin positive. In nonsustained virologic responders patients, 3 of 7 cryofibrinogen-positive patients (43%) at baseline became cryofibrinogen negative at the end of follow-up, whereas 10 of 15 cryofibrinogen-negative patients (67%) at baseline remained cryofibrinogen negative at the end of follow-up (P ⫽ .38). For patients who did not receive HCV treatment (n ⫽ 44), 4 of 16 cryofibrinogen-positive patients (25%) at baseline became cryofibrinogen negative at the end of follow-up, whereas 19 of 28 cryofibrinogen-negative patients (68%) at baseline remained cryofibrinogen negative at the end of follow-up (P ⫽ .01). Among cryofibrinogen-positive patients at baseline, 12 of 26 (46%) of those who received an HCV treatment became cryofibrinogen negative at the end of follow-up compared with 4 of 16 (25%) of those who did not receive antiviral drugs (P ⫽ .2). Respective rates for cryofibrinogen-negative patients at baseline were 22 of 30 (73%) and 19 of 28 (68%) (P ⫽ .7). In the entire cohort of HCV-infected patients, most cryofibrinogen-positive patients at baseline also were cryoglobulin positive (39/42 [93%]), and those who became cryofi-
Cryofibrinogen and Cryoglobulin Status in Hepatitis C Virus-Infected Patients at Baseline
CF positive, n (%) CF negative, n (%)
All Patients (N ⫽ 143)
HCV-Related Vasculitis (n ⫽ 57)
HCV-Infected Patients Without Vasculitis (n ⫽ 86)
CG⫹
CG⫺
CG⫹
CG⫺
CG⫹
CG⫺
47 (89%) 37 (41%)
6 (11%) 53 (59%)
27 (96%) 25 (86%)
1 (4%) 4 (14%)
20 (80%) 12 (20%)
5 (20%) 49 (80%)
HCV ⫽ hepatitis C virus; CF ⫽ cryofibrinogen.
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Figure 2 Spearman rank correlation between cryofibrinogen and cryoglobulin serum levels in the entire cohort of 143 HCV-infected patients (r2 ⫽ 0.764, P ⬍ .001) (left) and in 57 patients with HCV-related vasculitis (r2 ⫽ 0.759, P ⬍ .001) (right). CF ⫽ cryofibrinogen; CG ⫽ cryoglobulin.
Figure 3 Course of Cryofibrinogen Status in Patients Who Received Hepatitis C Virus Treatment.
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Table 3 Baseline Manifestations and Outcomes of Patients with Hepatitis C Virus-Related Vasculitis Who Received Hepatitis C Virus Treatment, According to the Cryofibrinogen/Cryoglobulin Status at Baseline P Cryoprotein Status at Baseline Cryofibrinogen Cryoglobulin Manifestations of the Vasculitis at Baseline Skin, n (%) Nerve Kidney Joints Response to Hepatitis C Virus Treatment Clinical response of the vasculitis Complete response Partial response Nonresponse Sustained virologic response
⫹ ⫹ 27/57 21/27 19/27 3/27 12/27
15/23 7/23 1/23 16/23
(47%)* (78%) (70%) (11%) (44%)
⫺ ⫹ 25/57 19/25 16/25 10/25 13/25
(44%)* (76%) (64%) (40%) (52%)
1.00 .77 .02 .78
(65%) (30%) (4%) (70%)
11/19 3/19 4/19 11/19
(58%) (16%) (21%) (58%)
.75 .30 .16 .52
*Among the patients with hepatitis C virus-related vasculitis, 4 were cryofibrinogen negative/cryoglobulin negative and 1 was cryofibrinogen positive/cryoglobulin negative.
brinogen negative at the end of follow-up also became cryoglobulin negative (13/14 [93%]), whereas 24 of 25 patients (96%) who remained cryofibrinogen positive also remained cryoglobulin positive. Among treated patients, 10 of 11 patients (91%) who became cryofibrinogen negative also became cryoglobulin negative, whereas 13 of 14 (93%) of those who remained cryofibrinogen positive also remained cryoglobulin positive.
Analysis of Patients with HCV-Related Vasculitis Fifty-seven patients had HCV-related vasculitis. The main clinical manifestations at baseline and the clinical and virologic outcomes according to cryoprotein status at baseline are detailed in Table 3. Clinical manifestations of HCVrelated vasculitis did not differ in the 27 cryofibrinogenpositive/cryoglobulin-positive patients compared with the 25 cryofibrinogen-negative/cryoglobulin-positive patients, except for a lower rate of renal involvement (40% vs 11%, respectively; P ⫽ .02). The rate of non-Hodgkin’s lymphoma was 8% (2/25) in cryofibrinogen-positive/cryoglobulin-positive patients versus 15% (4/27) in cryofibrinogen-negative/ cryoglobulin-positive patients (P ⫽ not significant). Five patients had clinical signs of HCV-related vasculitis without identification of cryoglobulin at baseline. Four patients were cryofibrinogen negative/cryoglobulin negative, and their vasculitis involved the skin (1), nerves (3), and kidneys (1). One patient who was cryofibrinogen positive/ cryoglobulin negative at baseline presented with peripheral nerve involvement. Forty-eight patients (84%) with vasculitis had a sufficient follow-up (mean 32.9 ⫾ 23.0 months [6-87]) for the analysis of clinical, immunologic, and virologic responses. There were no significant differences in clinical and virologic
responses between cryofibrinogen-positive/cryoglobulinpositive and cryofibrinogen-negative/cryoglobulin-positive patients. Among the cryofibrinogen-positive/cryoglobulinpositive patients at baseline, 10 became cryofibrinogen negative at the end of follow-up and also became cryoglobulin negative. Among the 14 patients who remained cryofibrinogen positive at the end of follow-up, only 1 patient became cryoglobulin negative. Fourteen patients had a renal involvement, with a biopsyproven membranoproliferative glomerulonephritis in 10 of 14 patients. In the 4 other patients, renal biopsy was not performed because a peripheral nerve biopsy already showed signs of cryoglobulin vasculitis. Among these patients, 3 of 14 were cryoglobulin positive/cryofibrinogen positive at baseline; 2 of 3 patients had a partial response and 1 of 3 patients had a complete response of the vasculitis after treatment. One patient was cryoglobulin negative/ cryofibrinogen negative at baseline and had a complete response of the vasculitis after treatment.
DISCUSSION Most HCV-infected patients (40%-60%) produce mixed cryoglobulin, a circulating serum cryoprotein composed of Igs and HCV-RNA particles. However, only a minority of HCV mixed cryoglobulin-positive patients will develop systemic vasculitis involving the skin, kidneys, and nervous system. The underlying mechanisms are not completely understood. Cryofibrinogen is another cryoprotein complex including fibrin, fibrinogen, factor VIII, and sometimes Ig. Cryofibrinogen can mimic some cryoglobulin manifestations, such as cold intolerance, purpura, skin necrosis, ulcers, and gangrene.17,22-24 Our study was designed to determine whether the presence of a cryofibrinogen may worsen mixed cryoglobulinemia-related vasculitis. In the
630 present study, we found that cryoproteins, including cryoglobulin and cryofibrinogen, are frequently found in the serum of HCV-infected patients. Cryofibrinogen was almost always found when cryoglobulin was present. Cryofibrinogen was more frequently found in patients with HCVrelated vasculitis than in those without vasculitis (49% vs 29%). In these patients, the presence of a cryofibrinogen was associated with a lower rate of renal involvement. Although antiviral therapy decreased the rate of cryofibrinogen positivity, the presence of a cryofibrinogen had no impact on clinical and virologic responses to antiviral therapy. The presence of a cryofibrinogen has been suggested as a marker of the severity of the underlying disease. A study of 284 patients with inflammatory bowel disease found that elevated cryofibrinogen was associated with a more severe disease and refractoriness to medication.29 Another study showed a 30% increased level of cryofibrinogen in 53 cases of nephrosis (24 cases) and acute and chronic renal failure (17 and 12 patients, respectively).30 A cryofibrinogen may cause intravascular coagulation in which fibrin can be deposited in the glomerular filters and may cause anuria, and in the renal vasculature where it may cause ischemic tubular necrosis. Other studies,22,28,31 however, have shown no correlation between levels of cryofibrinogen and disease intensity. In the present study, clinical signs and response to treatment were similar, regardless of the cryofibrinogen status at baseline. The only significant difference was that patients with vasculitis without cryofibrinogen had a higher rate of renal involvement. However, because there were few patients with renal involvement, definite conclusions cannot be drawn from our study. As observed with cryoglobulin,32,33 antiviral therapy decreased the incidence of cryofibrinogen positivity. This might be the result of the disappearance of cryoglobulin. The presence of a cryofibrinogen was strongly correlated with a positive cryoglobulin. Some 93% of cryofibrinogen-positive patients also were cryoglobulin positive, and cryofibrinogen levels were positively correlated with cryoglobulin levels. The same observation was made during follow-up. At the end of the study, a large majority of patients who became cryofibrinogen negative also were cryoglobulin negative. Taken together, these data support the hypothesis that cryofibrinogen may be linked to the presence of a cryoglobulin. The absence of a clinical and virologic impact of a cryofibrinogen in patients with vasculitis suggests that the presence of a cryofibrinogen may be a phenomenon linked to the presence of a cryoglobulin. We may ask if a cryofibrinogen is really a separate protein or a form of cryoglobulin that binds to fibrins products in the serum. Many arguments support that these are really 2 different cryoproteins. Only 56% of cryoglobulin-positive patients at baseline (and 52% of patients with HCV-related vasculitis) also were cryofibrinogen positive. Cryofibrinogen and cryoglobulin are clearly identified as 2 different cryoproteins with our
The American Journal of Medicine, Vol 121, No 7, July 2008 laboratory tests. A cryofibrinogen can be found without cryoglobulin with a particular clinical course.28
CONCLUSIONS Cryoproteins, including cryoglobulin and cryofibrinogen, are frequently found in the serum of HCV-infected patients. A cryofibrinogen is almost always found when a cryoglobulin is present. The presence of a cryofibrinogen has no impact on clinical and virologic responses to antiviral therapy. In such patients, positive cryofibrinogen status is closely related to the presence of a cryoglobulin at baseline and after antiviral therapy. Further studies are needed to determine whether the presence of a cryofibrinogen is associated with a lower rate of renal involvement in patients with mixed cryoglobulinemia-related vasculitis.
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