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. 2006 Nov;146(2):211–217. doi: 10.1111/j.1365-2249.2006.03196.x

Quantitative measurement of hepatitis C virus core antigen is affected by the presence of cryoglobulins

B-N Pham *, M Martinot-Peignoux †, M-P Ripault †, N Boyer †, V Levy ‡, P Marcellin †
PMCID: PMC1942051  PMID: 17034572

Abstract

Mixed cryoglobulinaemia is associated strikingly with HCV infection. The aim of this study was to assess whether the adherence to proper methods of collecting samples for cryoglobulin detection was critical or not on virological parameters in hepatitis C virus (HCV) patients. We studied 56 consecutive patients. Blood samples were collected using a conventional method and a blood collection method at 37°C adapted to cryoglobulin detection. HCV core antigen and HCV RNA were measured in sera and cryoglobulins issued from both blood collection methods. In cryoglobulin-positive patients, serum concentrations of HCV core antigen, but not that of HCV RNA, were significantly higher when a conventional method was used, compared to a blood collection method at 37°C (P = 0·001). In the cryoprecipitates, concentration of HCV core antigen was optimum when the blood collection method at 37°C, rather than the conventional method, was applied for cryoglobulin detection (P < 10−4). The recovery of HCV core antigen in the cryoprecipitate was improved when cryoglobulins were isolated using the blood collection method at 37°C rather than the conventional method (P < 0·001). HCV parameter measurements and cryoglobulin study should not be performed on the same serum samples due to the potential impact of blood collection methods on results.

Keywords: cryoglobulinaemia, cryoglobulins, HCV core antigen, HCV RNA, hepatitis C

Introduction

Cryoglobulinaemia is defined as the presence in the serum of immunoglobulins which precipitate at temperatures below 37°C and redissolve on rewarming. According to Brouet et al. [1], cryoglobulins can be subdivided into three types on the basis of immunochemical studies: type I consists of an isolated monoclonal immunoglobulin; type II consists of a mixture of monoclonal immunoglobulin and polyclonal immunoglobulins; and type III consists of a mixture of polyclonal immunoglobulins. Type I cryoglobulins are associated with multiple myeloma, Waldenström's macroglobulinaemia and lymphoproliferative disorders. Type II and Type III cryoglobulins are associated with viral, bacterial, parasitic infections and numerous systemic diseases. Type II and type III cryoglobulins are mixed cryoglobulinaemia, the common feature of which is rheumatoid factor activity. Clinical manifestations associated with mixed cryoglobulinaemia consist of a triad of palpable purpura, arthralgias and weakness [2]. Because of its clinical polymorphism and even subclinical manifestations, the frequency of mixed cryoglobulinaemia can be underestimated. In addition, the detection of cryoglobulins in serum requires some simple but important precautions: it is necessary to keep blood samples at about 37°C, and to clot and centrifuge it at the same temperature, after which the serum should be refrigerated at 4°C for 7 days.

Hepatitis C virus (HCV) is responsible for a large proportion of viral hepatitis worldwide. Acute hepatitis is frequently asymptomatic; however, development of chronic liver disease occurs in at least 70% of infected patients. It is now widely accepted that mixed cryoglobulinaemia is associated with HCV infection. Several studies have reported a high prevalence of cryoglobulinaemia in patients with chronic hepatitis C [3–7]. Conversely, the prevalence of anti-HCV antibodies in patients with mixed cryoglobulinaemia range from 70% to 100%, suggesting an important role for HCV in the pathogenesis of mixed cryoglobulinaemia [8–10].

In newly diagnosed patients, HCV status is assessed on virological parameters such as genotype and viraemia. HCV genotyping, HCV RNA detection and viral load quantification are based on molecular biology techniques. Recently, a commercial assay for the detection and quantification of the core antigen of HCV has been developed by Ortho Clinical Diagnostics (Raritan, NJ, USA), providing an attractive alternative to HCV RNA testing [11–14]. In newly diagnosed patients with hepatitis C infection, the presence of cryoglobulins is not known at the time of diagnosis. The aim of this study was to assess whether the presence of cryoglobulins interfere in the determination of HCV status and whether adherence to the proper method of collecting and processing samples for cryoglobulin detection is critical or not on virological parameters in patients with chronic hepatitis C.

Patients and methods

Patients

This prospective study included 61 patients. Fifty-six patients had chronic hepatitis C diagnosed on the basis of clinical data, aminotransferase levels, serological markers (see below) and histological data. All these patients had anti-HCV antibodies. They were negative for hepatitis B surface antigen, anti-HIV antibodies and anti-mitochondrial and liver–kidney microsomes antibodies. Other causes of chronic liver disease (Wilson's disease, alcoholism, hepatotoxic drugs or α1-anti-trypsin deficiency) were excluded. None of these patients had received anti-viral treatment before inclusion. All patients were investigated for the presence of cryoglobulins.

Five control patients with lymphoproliferative disorder (multiple myeloma) associated with cryoglobulinaemia were included. In these patients, serological viral markers were not detected. Serum HCV RNA was not detected by reverse transcription–polymerase chain reaction (RT–PCR). Other types of liver disease were excluded.

This study was carried out according to the ethical guidelines of the 1975 Declaration of Helsinki.

Methods

Screening procedure

All patients had been tested previously for antibodies to HCV using third-generation enzyme-linked immunosorbent assays (ELISA) (Abbott Laboratories, Chicago, IL, USA and Biorad, Marnes-la Coquette, France). The other virological markers [hepatitis B surface antigen (HBsAg), antibodies to HBsAg, immunoglobulin M (IgM) to HBcAg, IgM to hepatitis A virus (HAV), antibodies to HCV] were determined with commercially available kits (Abbott Laboratories). Anti-nuclear antibodies and anti-tissue antibodies were investigated by indirect immunofluorescence.

For each patient, blood samples were collected using two different methods at the time of sampling in order to compare the impact of cryoglobulins on virological results. This was accomplished by having the blood drawn freshly into two series of tubes. The first series used prewarmed (37°C) tubes. This first series of samples was allowed to clot at 37°C in a water bath for 1 h and then separated immediately by centrifugation at 1500 g for 10 min. The second series used normal tubes. This second series of tubes was allowed to clot at room temperature for 1 h and then separated immediately by centrifugation at 1500 g for 10 min. After centrifugation, different aliquots from the first and the second series of tubes were prepared for the study and cryoglobulin determination of serum virological markers. When issued from clotting at 37°C, samples were labelled ‘37°C’ samples. When issued from clotting at room temperature, samples were labelled ‘RT’ samples.

Cryoglobulin determination

After clotting (37°C or room temperature conditions) and centrifugation, freshly collected serum was placed at 4°C for 7 days after addition of antiseptic. In order to prevent bacterial contamination that could be misinterpreted as the presence of cryoglobulins, antiseptic is used. When cryoglobulins were detected visually, cryoprecipitates were separated from the remaining surpernatants by centrifugation at 3000 g for 30 min at 4°C. Cryoprecipitates were stored immediately at −80°C for HCV antigen quantification and HCV RNA analysis as serum samples.

HCV core antigen

HCV core antigen was quantified on 37°C and RT samples of serum. When cryoglobulins were detected, HCV core antigen quantification was assessed on cryoprecipitates isolated from 37°C and RT serum samples.

HCV core antigen quantification was determined using the Ortho track-C assay (Ortho Clinical Diagnostics). This immunoenzymatic test, designed to detect the HCV core antigen, involves a pretreatment step to dissociate immune complexes and lyse viral particles. Free core antigen is captured with murine anti-core antigen monoclonal antibodies. Briefly, 100 µl of the serum sample was mixed with 50 µl of a pretreatment solution. After incubation at 56°C for 30 min, 100 µl of the pretreated solution was added to a well coated with monoclonal antibodies against the HCV core antigen and filled with 100 µl of reaction buffer. The mixture was incubated with agitation for 60 min at room temperature and then washed with buffer. Horseradish peroxidase-conjugated reagent was added to the well and incubated for 30 min at room temperature. The microwells were washed again, followed by the addition of 200 µl of o-phenylenediamine substrate solution. After incubation for 30 min at room temperature in the dark, the reaction was stopped with the addition of 50 µl of 5 N H2SO4. The optical density of the solution in the microwells was determined at 490 nm with a 650-nm reference. The concentration of the HCV core antigen was determined according to a standard curve obtained with the calibrators and expressed in pg/ml. A result of > 0 pg/ml indicated the presence of HCV core antigen.

HCV RNA detection

HCV RNA was detected on 37°C and RT samples of serum by transcription-mediated amplification (TMA) (Bayer Diagnostics, Emeryville, CA, USA). The limit of detection of this assay was 50 copies/ml (10 IU/ml).

HCV RNA quantification

HCV RNA was quantified on 37°C and RT samples of serum. When cryoglobulins were detected, HCV RNA quantification was assessed on cryoprecipitates isolated from 37°C and RT serum samples. HCV RNA was quantified using the bDNA signal amplification assay (Versant HCV RNA 3·0 assay; Bayer Diagnostics). The limit of detection of the latter assay was 2·5 × 103 copies/ml (521 IU/ml).

Statistical analysis

Comparisons were made using Wilcoxon's signed-rank test.

Results

Of the 56 patients screened for hepatitis C, 34 patients (60·7%) had chronic hepatitis C with cryoglobulinaemia, whereas 22 patients (39·2%) had chronic hepatitis C without cryoglobulinaemia. In all patients, HCV core antigen and HCV RNA were measured in 37°C and RT serum samples. In HCV patients with cryoglobulinaemia, HCV core antigen and HCV RNA were measured in cryoprecipitates.

Patients with cryoglobulinaemia

The 34 patients with cryoglobulinaemia comprised 19 men and 15 women. The HCV genotype was 1 in 10 patients, 2 in six patients, 3 in seven patients, 4 in three patients and 5 in one patient.

Serum sample analysis

HCV core antigen

HCV core antigen could be measured in 31 of 34 patients (Fig. 1). For the 37°C serum samples, HCV core antigen concentrations ranged from 1·8 pg/ml to 1298 pg/ml (median 70·8 pg/ml). For the RT serum samples, HCV core antigen concentrations ranged from 1·9 pg/ml to 1382 pg/ml (median 79 pg/ml). HCV core antigen concentrations were significantly higher in RT serum samples than in 37°C serum samples (P = 0·001). However, no HCV core antigen-positive RT serum samples displayed negative results when measured in 37°C serum samples.

Fig. 1.

Fig. 1

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Measurement of serum hepatitis C virus (HCV) core antigen in cryoglobulin-positive patients with HCV infection. Serum was collected using a conventional method of blood collection and a blood collection method at 37°C adapted to cryoglobulin detection. HCV core antigen concentrations were significantly higher with a conventional method of blood collection than with a blood collection method at 37°C (P = 0·001).

HCV RNA

HCV RNA could be measured in 33 of 34 patients. For the 37°C serum samples, HCV RNA concentrations ranged from 37 724 copies/ml to 24 814 670 copies/ml (median 1817 407 copies/ml). For the RT serum samples, HCV RNA concentrations ranged from 30 452 copies/ml to 38 264 284 copies/ml (median 1351 976 copies/ml). The difference was not significant.

Cryoglobulin analysis

HCV core antigen

HCV core antigen could be measured in all 34 purified cryoglobulins (Fig. 2). When cryoglobulins were purified from 37°C serum samples, HCV core antigen concentrations ranged from 2·7 pg/ml to 3805 pg/ml (median 332 pg/ml). When cryoglobulins were purified from RT serum samples, HCV core antigen concentrations ranged from 2·6 pg/ml to 3540 pg/ml (median 178 pg/ml). HCV core antigen concentrations were significantly lower than in cryoglobulins purified from 37°C serum samples (P < 10−4).

Fig. 2.

Fig. 2

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Measurement of hepatitis C virus (HCV) core antigen in cryoglobulins issued from serum samples obtained with a conventional method of blood collection and a blood collection method at 37°C adapted to cryoglobulin detection. In cryoprecipitates isolated from serum samples obtained with a conventional method of blood collection, HCV core antigen concentrations were significantly lower than in cryoprecipitates isolated from serum samples obtained with the blood collection method at 37°C (P < 10−4). A logarithmic scale was used for values of HCV core antigen concentrations.

Serum/cryoglobulin HCV core antigen ratios were calculated to assess enrichment of HCV core antigen in cryoglobulins (Fig. 3). When cryoglobulins were purified from 37°C serum samples, the ratios ranged from 0·10 to 3·42 (median 0·3). When cryoglobulins were purified from RT serum samples, the ratios ranged from 0·21 to 7·6 (median 0·5). Ratios were significantly lower when cryoglobulins were purified from 37°C serum than when cryoglobulins were purified from RT serum samples (P < 0·001).

Fig. 3.

Fig. 3

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Serum/cryoglobulin hepatitis C virus (HCV) core antigen ratios. The recovery of HCV core antigen in the cryoprecipitate was improved when cryoglobulins were isolated using the blood collection method at 37°C rather than the conventional method (P < 0·001).

HCV RNA

HCV RNA could not be measured in purified cryoglobulins because the bDNA signal amplification assay gave false positive results when performed on cryoprecipates from HCV negative patients (see Control patients section).

Patients without cryoglobulinaemia

The 22 chronic hepatits C patients without cryoglobulinaemia comprised nine men and 13 women. The HCV genotype was 1 in eight patients, 2 in six patients and 4 in two patients.

HCV core antigen

HCV core antigen could be measured in 21 of the 22 patients. For the 37°C serum samples, HCV core antigen concentrations ranged from 1·8 pg/ml to 626 pg/ml (median 67·2 pg/ml). For the RT serum samples, HCV core antigen concentrations ranged from 1·9 pg/ml to 550 pg/ml (median 73·6 pg/ml). The difference was not significant.

HCV RNA

HCV RNA could be measured in 21 of 22 patients. For the 37°C serum samples, HCV RNA concentrations ranged from 81 253 copies/ml to 34 798 708 copies/ml (median 4389 548 copies/m). For the RT serum samples, HCV RNA concentrations ranged from 147 967 copies/ml to 30 986 146 copies/ml (median 3566 657 copies/ml). The difference was not significant.

Control patients

Control patients comprised six patients with cryoglobulinaemia related to their malignant haematological disease. These patients were negative for anti-HCV antibodies. HCV core antigen was measured in the six purified cryoglobulins. The samples were all negative. HCV RNA was quantified in the six purified cryoglobulins. The samples displayed false positive results (3200–10 000 copies/ml).

Discussion

Mixed cryoglobulinaemia is associated strikingly with HCV infection. In the present study, we report the detection of cryoglobulins in 60·7% of patients with chronic hepatitis C when the correct method of collecting and processing samples for cryoglobulin detection is used. The adherence, or not, to a meticulous method may partly explain the prevalence of cryoglobulinaemia in HCV infection, ranging from 19% to 68% [3–7]. Indeed, a correct method for cryoglobulin detection must control numerous parameters, such as the use of prewarmed tubes, blood taken from a fasting patient, addition of antiseptic in the serum and prompt transport to the laboratory, because delay in the transport of the sample before processing leads to the loss of cryoprecipitable substances in the clot.

With respect to viral factors, no predominance of a specific HCV genotype was found in cryoglobulin-positive patients with HCV infection when compared to cryoglobulin-negative patients with HCV infection, in accordance with previous studies [15,16]. With regard to HCV viraemia, the latter was assessed systematically by HCV RNA and HCV core antigen quantification in our study. We found a high prevalence of HCV viraemia among cryoglobulin-positive and cryoglobulin-negative patients (97% and 95%, respectively) when HCV RNA quantification was used as the viraemia marker. Despite a good correlation between the serum measurements of HCV core antigen and HCV RNA, HCV core antigen measurement was a less sensitive diagnostic assay for HCV viraemia than HCV RNA quantified by branched-DNA assay, as described previously [17,18].

This study demonstrates that in cryoglobulin-positive patients with HCV infection serum measurement of HCV core antigen, but not that of HCV RNA, is affected by the presence of cryoglobulins. As cryoglobulins need a reliable method to be detected correctly, our study focused on the first steps of the method to collect samples to assess whether these precise technical conditions for efficient cryoglobulin detection may or may not affect HCV parameter measurements. We have shown that HCV core antigen concentrations were significantly higher when a conventional method of blood collection for cryoglobulin detection was used (clotting at room temperature for 1 h) compared to that obtained with a precise method of blood collection (blood drawn in prewarmed tubes and clotting at 37°C for 1 h) (P = 0·001). The drop in HCV core antigen concentrations when blood was allowed to clot at 37°C for 1 h was not due to a potential degradation of HCV core antigen at 37°C, as we demonstrated that there was no significant difference when HCV core antigen concentrations were measured with respect to both blood collection methods in cryoglobulin-negative patients with HCV infection. The drop of HCV core antigen concentrations when blood was allowed to clot at 37°C for 1 h was not due to a loss of HCV particles, as we demonstrated that there was no significant difference when HCV RNA concentrations were measured with respect to both blood collection methods in HCV infected patients. HCV may circulate in the blood in two forms: free HCV and the virus complexed to antibodies [19–21]. The tendency of antibodies to form stable complexes with the antigen is influenced by many physicochemical conditions, such as temperature. In many cases, antibody affinity may be optimum at 37°C. On the whole, our data suggest that the blood collection method at 37°C may influence interaction of the HCV core antigen with anti-HCV core antibodies, when the latter have a distinctive feature to precipitate when cold. The first step of the HCV core antigen assay, a pretreatment step to dissociate immune complexes and lyse viral particles, may be insufficient to dissociate these complexes correctly. This hypothesis has been posited in a previous study [22]. In addition, the differences of IgG antibody avidity in HCV infection has been reported [23]. Because cryoglobulinaemia is associated strikingly with HCV infection, physicians must be aware that viral parameter measurements and cryoglobulin study may not be performed on the same serum samples, due to the potential impact of the blood collection methods on results. In addition, future assays based on concomitant HCV antigen and anti-HCV antibody detection should be validated in cryoglobulin-positive patients.

In cryoglobulin-positive patients with HCV infection, HCV particles are concentrated in the cryoprecipitate [20,24]. In this study, we found that concentration of HCV core antigen in the cryoprecipitates was optimum when the blood collection method was applied at 37°C for cryoglobulin detection. When cryoprecipitates were isolated from serum samples obtained with a conventional method of blood collection, HCV core antigen concentrations were significantly lower than in cryoprecipitates isolated from serum samples obtained with the 37°C blood collection method (P < 10−4). In addition, the recovery of HCV core antigen in the cryoprecipitate was found to be improved significantly when cryoglobulins were isolated from serum samples obtained with the 37°C blood collection method when compared to cryoglobulins isolated from serum samples obtained with a conventional method of blood collection (P < 0·001). Whereas HCV core antigen could be quantified in all the cryoprecipitates, HCV RNA quantification could not be performed in parallel to HCV core antigen measurement because the high concentration of immunoglobulins forming the cryoglobulin induced technical interference in the branched-DNA assay. The increased concentration of HCV core antigen in the cryoprecipitate was related to the increased isolation of cryoglobulins from serum samples obtained with the 37°C blood collection method. After purification, the cryoglobulin amount can be determined using different methods. However, no method is accurate enough to detect a difference in cryoglobulin amount related to the blood collection method. The real mechanism(s) of cryoprecipitation remains obscure. In our study, we have shown that there was a selective concentration of HCV core antigen in cryoglobulins, in accordance with previous data. In addition, previous studies have reported the presence of anti-HCV antibodies in cryoglobulins [24–26]. On the whole, these data on HCV immune complexes concentrated in the cryoprecipitate suggest a role of HCV in the pathogenesis of mixed cryoglobulinaemia.

Acknowledgments

The authors thank J. H. M. Cohen for his critical advice, V. Esnault for her help in collecting blood samples, E. Nobile (Ortho-Clinical Diagnostics) for providing the HCV core antigen kits and F. Huisse (Bayer Diagnostics) for providing the bDNA 3·0 kits.

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