Piermario Bellavita

Interferon Alfa-2a Therapy in Cryoglobulinemia Associated with Hepatitis C Virus

BACKGROUND Essential mixed cryoglobulinemia is frequently associated with hepatitis C virus (HCV) infection. A beneficial effect of interferon alfa therapy has been reported, but we do not know whether the antiviral activity of the drug affects the clinical and biochemical manifestations of disease. METHODS In a prospective randomized, controlled trial, we studied 53 patients with HCV-associated type II cryoglobulinemia. A group of 27 patients received recombinant interferon alfa-2a thrice weekly at a dose of 1.5 million units for a week and then 3 million units thrice weekly for the following 23 weeks. The 26 control patients did not receive anything apart from previously prescribed treatments. All patients were then followed for an additional 24 to 48 weeks. RESULTS Interferon was usually well tolerated, but it was permanently discontinued in two patients because of atrial fibrillation and depression. Two of the 26 patients in the control group were lost to follow-up. After the treatment period, serum HCV RNA was undetectable in 15 of the remaining 25 patients who received interferon alfa-2a, but in none of the controls. In comparison with the control group, the 15 patients with undetectable levels of HCV RNA in serum had significant improvement in cutaneous vasculitis (P = 0.04) and significant decreases in serum levels of anti-HCV-antibody activity (P = 0.007), cryoglobulins (P = 0.002), IgM (P = 0.002), rheumatoid factor (P = 0.001), and creatinine (P = 0.006). After treatment with interferon alfa-2a was discontinued, viremia and cryoglobulinemia recurred in all 15 HCV RNA-negative patients. On resumption of treatment, three of four patients had a virologic, clinical, and biochemical response. CONCLUSIONS The therapeutic efficacy of interferon alfa-2a in HCV-associated cryoglobulinemia is closely related to its antiviral activity, thus supporting the idea that HCV infection may be a cause of this disease.

Hepatitis C virus infection in patients with essential mixed cryoglobulinemia

OBJECTIVE To study the association between hepatitis C virus (HCV) infection and essential mixed cryoglobulinemia. SETTING Wards and clinics of the Ospedali Riuniti di Bergamo and Ospedale di Treviglio e Caravaggio, Italy. PATIENTS Fifty-one patients with essential mixed cryoglobulinemia associated with glomerulonephritis and 45 controls with noncryoglobulinemic glomerulopathies. MEASUREMENTS Antibodies to hepatitis C virus (anti-HCV) in sera from patients with essential mixed cryoglobulinemia and from controls, using two enzyme-linked immunosorbent assays (c100 ELISA and c22/c200 ELISA) and a recombinant immunoblot assay (4-RIBA); cryoprecipitate anti-HCV before and after use of dithiothreitol, a substance able to destroy IgM antibodies with rheumatoid factor activity, in patients with essential mixed cryoglobulinemia; serum HCV RNA by polymerase chain reaction in patients with essential mixed cryoglobulinemia. RESULTS In patients with essential mixed cryoglobulinemia, the c22/c200 ELISA detected anti-HCV in 98% of serum samples (95% CI, 90% to 100%), whereas the rate of reactivity remained at 2% (CI, 0% to 12%) in the control group (P less than 0.0001). These results were confirmed by the 4-RIBA in 66% of patients with essential mixed cryoglobulinemia. The study of cryoprecipitate by c100 ELISA showed anti-HCV in 41% (Cl, 28% to 56%) of patients. After dithiothreitol, the rate of reactivity increased to 94% (CI, 84% to 99%; P less than 0.0001 by the McNemar paired chi-square test), suggesting that the elimination of rheumatoid factor leads to unmasking of anti-HCV in cryoprecipitate. Polymerase chain reaction detected HCV RNA in 13 of 16 sera from patients with essential mixed cryoglobulinemia. CONCLUSIONS The extremely high prevalence of anti-HCV in serum and cryoprecipitate along with the frequently associated serum HCV RNA suggests a close relation between essential mixed cryoglobulinemia and chronic HCV infection.

Negative selection of peripheral blood stem cells to support a tandem autologous transplantation programme in multiple myeloma

Summary. We recently described a two‐step negative selection procedure whereby peripheral blood stem cells (PBSCs) were efficiently purged of contaminating neoplastic cells by a combination of monoclonal antibodies. Here, we report 60 newly diagnosed multiple myeloma (MM) patients treated with a double transplant programme and randomized to receive either unmanipulated or in vitro purged PBSCs. We demonstrated that this technique is feasible and safe without significant loss of either CD34+ or CD3+ cells. Haematological engraftment and immunological reconstitution were rapid without treatment‐related mortality. Using polymerase chain reaction (PCR), we compared the level of minimal residual disease (MRD) in PBSC before and after in vitro purging and in vivo after transplant. A median of one tumour cell per 102 normal cells (range 101−105) was seen in the unmanipulated aphereses with a 3–4 log reduction after manipulation in vitro. However, despite this tumour debulking, all patients remained PCR positive in vivo. At 3 years, the estimated event‐free survival was 40% in the control arm and 72% in the experimental arm (P = 0·05), whereas the estimated overall survival was 83% in both arms. This suggests that autologous transplantation using efficiently purged PBSCs can be performed safely, but confirms the need for innovative protocols for MRD eradication in vivo.

HEPATITIS C VIRUS IN RENAL TISSUE OF PATIENTS WITH GLOMERULONEPHRITIS

Dr. Piermario Bellavita, Department of Immunohaematology, Ospedali Riuniti, Largo Barozzi 1, I-24128 Bergamo (Italy) Dear Sir, We have recently found the presence of hepatitis C virus (HCV) markers in sera and cryoprecipitates from most patients with cryoglobulinemic glomerulonephritis [1]. Moreover, the association between renal disease and HCV infection has also been reported in some cases of membranoprolifera-tive glomerulonephritis without detectable serum cryoglobulins [2]. However, the causal role of HCV in producing histological lesions and clinical features of cryoglobulinemic and noncryoglobulmemic glomerulonephritides has not been demonstrated. Very recently, Doutrelepont et al. [3] described a patient with membranoproliferative glomelurone-phritis associated with mixed cryoglobuline-mia and HCV infection. The detection of HCV RNA by the polymerase chain reaction (PCR) in urine and renal tissue from this patient led the authors to infer that HCV was responsible for the glomerulonephritis. One can argue that several viruses can easily enter the urine during the viraemic stage even in the absence of obvious renal lesions, and that detection of HCV in renal tissue by PCR does not necessarily prove the involvement of this agent in the pathogenesis of the renal disease [4]. Indeed, PCR is a highly sensitive test potentially able to reveal the presence of a single HCV RNA molecule in the infected blood of a normal kidney specimen. In order to assess the possibility of an unspecific finding, we tested the renal tissue of 13 anti-HCV positive patients for HCV RNA; 10 of these patients had various forms of glomerulopathy and 3 had histologically normal kidneys. HCV RNA was extracted from paraffin-embedded kidney specimens obtained by renal biopsy in 7 cases and by autopsy in 6. PCR was performed with primers derived from the 5’-noncoding, highly conserved region of the HCV genome, as previously described [5]. We found HCV RNA in renal tissue from 4 of 5 patients with cryoglobulinemic glomerulonephritis, 3 of 5 with noncryoglobulmemic glomerulopathy, and 2 of 3 with normal kidney. Our findings indicate that the detection of HCV RNA by PCR in renal tissue of patients with glomerular disease cannot be viewed as evidence of a cause and effect relationship between HCV and renal lesions. Nevertheless, these results do by no means exclude that HCV may be the major aetiologic agent of cryoglobulinemic glomerulonephritis. References

GB virus C Infection in patients with type II mixed cryoglobulinemia

Type II mixed cryoglobulinemia is characterized by purpura, arthralgias, weakness, glomerulonephritis, peripheral neuropathy, and other manifestations of systemic vasculitis. Its pathogenesis probably depends on cryoprecipitable circulating immunocomplexes consisting of polyclonal IgG and monoclonal IgM, the latter having rheumatoid factor activity [1-3]. Although mixed cryoglobulinemia is commonly associated with hepatitis C virus (HCV) [4-8], a variable proportion of cryoglobulinemic patients does not have markers of infection with any known viral agent. We investigated the possible role of GB virus C (GBV-C), a newly discovered RNA virus that shares some sequence homology with HCV [9-11], in type II mixed cryoglobulinemia. Methods We studied 58 patients with type II mixed cryoglobulinemia that was not associated with hematologic malignant conditions, autoimmune disorders, or infectious diseases other than chronic viral hepatitis. One hundred forty-five volunteer blood donors served as healthy controls. We also studied a second control group that included 20 noncryoglobulinemic blood donors who were age-matched with cryoglobulinemic patients and were positive for antibodies to HCV. All patients were tested for liver and kidney function according to standard methods. The serum concentration of immunoglobulins, rheumatoid factor activity, and C3 and C4 components were measured by using routine nephelometric assays. Cryoglobulins were measured as the protein concentration of isolated cryoprecipitate and were characterized by using standard immunoelectrophoresis and immunofixation procedures. Antibodies to HCV were measured with a second-generation enzyme-linked immunosorbent assay (Abbott Laboratories, North Chicago, Illinois). To search for enrichment of antibodies to HCV in cryoglobulins, the isolated and washed cryoprecipitate was resuspended in a volume of phosphate-buffered saline equal to the volume of the original serum. The activity of the antibodies to HCV per mg of IgG was subsequently measured, as reported elsewhere [12], in serial fivefold dilutions of both cryoprecipitate and supernatant. Serum specimens were tested for HCV RNA with a reverse-transcription polymerase chain reaction (PCR) assay, as described elsewhere [5]. The HCV viral load was measured in both cryoprecipitate and supernatant by using a quantitative reverse-transcription PCR assay (Amplicor HCV Monitor, Roche Diagnostic Systems, Inc., Branchburg, New Jersey), according to the manufacturers instructions. The GBV-C genome was detected by using a reverse-transcription PCR method developed in our laboratory [13]. Total RNA was extracted from 100 L of serum and was reverse transcribed by using random esanucleotides as primers. Complementary DNA was amplified through 45 cycles by using a pair of primers spanning a 161-base pair sequence in the conserved GBV-C helicase region. An aliquot of the PCR product was hybridized to an internal probe with a DNA enzyme immunoassay [14]. The optical densities of this immunoassay were used to measure GBV-C RNA levels in serial tenfold dilutions of cryoprecipitate-supernatant pairs. Twenty-seven cryoglobulinemic patients received recombinant interferon-alpha2b, and 8 received human lymphoblastoid interferon-, 4.5 to 6 million U three times weekly for 6 months and then 3 million U three times weekly for 6 to 12 months. For our purposes, virologic response was defined as the disappearance from serum of GBV-C RNA, HCV RNA, or both as a result of interferon- therapy. A clear alleviation of the signs and symptoms of cryoglobulinemia, along with a reduction of 50% or more in serum cryoglobulin levels, was considered a clinical response. Statistical analyses were done by using the two-sample Wilcoxon test, the chi-square test, or the Wilcoxon matched-pairs signed-rank test, as appropriate. All P values are two tailed, and statistical significance was set at the 0.05 level. Results We detected GBV-C RNA in 23 of the 58 patients with type II mixed cryoglobulinemia (40%); 20 of the 23 were also infected with HCV. We found HCV RNA alone in 28 patients. We detected GBV-C RNA in 1 of 145 unselected blood donors; thus, the prevalence of this virus was much lower among unselected blood donors than among cryoglobulinemic patients (0.7% compared with 40%; P < 0.001). Similarly, only a small proportion of blood donors was positive for antibodies to HCV compared with cryoglobulinemic patients (2.7% compared with 81%; P < 0.001). In contrast, the prevalence of GBV-C infection was similar in cryoglobulinemic patients positive for antibodies to HCV and age-matched blood donors positive for these antibodies (43% compared with 35%; difference, 8% [95% CI, 18% to 33%]; P > 0.2). As indicated above, 20 of 58 cryoglobulinemic patients (34%) were co-infected with GBV-C and HCV. No obvious difference was found when the demographic, clinical, and laboratory features of these patients were compared with those of patients infected with HCV alone. However, the patients with GBV-C and HCV co-infection tended to have slightly higher levels of cryoglobulins (P = 0.18) and rheumatoid factor activity (P > 0.2) and lower concentrations of C4 (P = 0.05). We detected GBV-C RNA alone in the serum specimens of three cryoglobulinemic patients who seemed to be clinically similar in all respects to patients infected with HCV and patients without demonstrable HCV infection. We measured the concentration of viral markers in samples of cryoprecipitate and supernatant from 10 patients. Results in patients who were co-infected with HCV and GBV-C are shown in the (Table 1). The concentration of antibodies to HCV, as expressed by the antibody activity per mg of IgG, was clearly higher in the cryoprecipitate than in the supernatant (P = 0.022). Similarly, the HCV RNA level was consistently greater in the cryoprecipitates than in the supernatants obtained from the same patients (P = 0.036). In contrast, GBV-C RNA levels seemed to be lower in cryoprecipitate than in supernatant in 4 of the 5 patients studied in this group (P > 0.2). Finally, GBV-C RNA was undetectable in all three cryoprecipitate specimens obtained from patients infected with GBV-C alone. Table 1. Quantitative Evaluation of Antibodies to Hepatitis C Virus, Hepatitis C Virus RNA, and GB Virus C Supernatant from Cryoglobulinemic Patients Co-Infected with Hepatitis C Virus and GB Virus C* Twenty-eight of 35 patients receiving interferon- responded by clearing circulating GBV-C, HCV, or both. The clinical condition did not improve in either of the two patients in whom GBV-C RNA but not HCV RNA disappeared from serum. In contrast, a clinical response was evident in 5 of 6 patients who cleared HCV RNA alone and 6 of 7 patients who cleared both HCV RNA and GBV-C RNA. Of the 18 patients with isolated HCV infection, 13 had both a virologic and a clinical response. Discussion Infection with GBV-C or its homologue, hepatitis G virus, has been demonstrated in many patients with such conditions as acute and chronic hepatitis, intravenous drug use, hemophilia, aplastic anemia, and maintenance hemodialysis [9-11, 15-18]. However, the pathogenic role of these viruses has not been established. We show that GBV-C infection, usually associated with HCV, is frequently found in patients with type II mixed cryoglobulinemia. It is interesting that the prevalence of GBV-C infection is equally high in healthy blood donors and cryoglobulinemic patients who are positive for antibodies to HCV. This suggests a common route of transmission for both viruses and may be taken as evidence against a causal role for GBV-C in type II mixed cryoglobulinemia. Moreover, because risk factors for parenteral exposure were uncommon in our patients with cryoglobulinemia, it is likely that most of these patients had community-acquired infection with cotransmitted GBV-C and HCV. No clinical or biochemical characteristic clearly distinguished the cryoglobulinemic patients with GBV-C infection from those who were not infected. Nonetheless, the tendency of cryoglobulinemic patients with GBV-C and HCV co-infection to have more evident immunologic abnormalities than patients infected by HCV alone suggests a pathogenic role for GBV-C. This possibility is also supported by the fact that GBV-C was the only agent identified in three cryoglobulinemic patients. Because mixed cryoglobulinemia is commonly considered to be a vasculitic process mediated by cryoprecipitable circulating immunocomplexes, several authors have sought markers of HCV infection in cryoprecipitate; some have found increased concentrations of antibodies to HCV, HCV RNA, or both [5-8]. In our patients with GBV-C and HCV co-infection, we found that concentrations of both antibodies to HCV and HCV RNA were consistently higher in cryoprecipitates than in supernatants, whereas levels of GBV-C RNA were lower in most cryoprecipitates obtained from the same patients. Moreover, GBV-C RNA was not detected in the cryoprecipitates of any of the three patients with isolated GBV-C infection. Recent therapeutic trials [12, 19] indicate that the effectiveness of interferon- in HCV-associated mixed cryoglobulinemia is related to the drugs antiviral activity. We were able to study the effects of interferon- in patients co-infected with GBV-C and HCV. In these patients, we found that response with respect to GBV-C infection, expressed by the disappearance of viral genome from serum, was as frequent as response with respect to HCV infection. However, our most important finding was that clinical response was related to the virologic response with respect to HCV but not GBV-C infection. Thus, our results suggest that GBV-C does not have a primary role in the pathogenesis of type II mixed cryoglobulinemia in patients co-infected with GBV-C and HCV. Although it seems unlikely, the situation could be different in cryoglobulinemic patients with isolated GBV-C infection. These patients formed a small

Comparison of performance of six different cell separators in collecting peripheral blood mononuclear cells

We compared the efficacy of six different cell separators in collecting peripheral mononuclear cells to be used for autologous or homologous peripheral stem cell transplantation. The product obtained with the Dideco Vivacell cell separator showed a low percentage of mononuclear cells (38%) in the final product and a high platelet efficiency (38%). The Baxter CS3000 Plus cell separator required the longest time to load and prime the kit (18 min), it showed a high MNC efficiency (68%), with the highest percentage of MNC in the final product, the highest platelet efficiency (45%), a low red blood cell contamination in the final product (2.7 mL), the highest extracorporeal volume (450 mL) and a high percentage of technical failures (15%). The product obtained with the Fresenius AS104 cell separator with P1Y kit showed the highest final volume (297 mL), the lowest platelet efficiency (12%) and the lowest extracorporeal volume (230 mL). The same cell separator with C4Y kit showed a lower MNC efficiency (52 vs 60%) and a higher percentage of MNC in final product (63 vs 41%). The platelet contamination in final product was the lowest (18 x 10(9)/100 mL). The Haemonetics MCS3p cell separator required the lowest time to load and prime the kit (5 min), it showed the highest MNC efficiency (71%). The blood volume processed per hour (1328 mL) and the percentage of MNC in final product was lowest (32%), the extracorporeal volume (450 mL) was the highest. The Cobe Spectra cell separator allowed to process the highest blood volume per hour (3383 mL) and the final product had the lowest red blood cell contamination (2.3 mL/100 mL). The Dideco Excel cell separator required the longest time to load and prime the kit (18 min), the lowest MNC efficiency (38%), the highest platelet contamination in final product. Furthermore this machine showed the highest percentage of technical failure (20%). None of the six instruments have all the required preconditions and the ideal cell separator for peripheral stem cell apheresis at present is not available on the market.

A Simple Mathematical Approach to Calculate Blood Loss in Radical Prostatectomy

Introduction: The aim of this study was to apply a simple mathematical approach to calculate blood loss in 126 patients undergoing radical retropubic prostatectomy (RRP). Materials and Methods: Perioperative red blood cell loss (RBCL) was estimated by adding the difference in circulating red blood cells from before to after surgery to the allogeneic red blood cells transfused in the same period. Results: Mean preoperative hematocrit was 45 ± 4% and mean perioperative RBCL was 574 ± 297 ml, corresponding to a mean equivalent whole blood loss (WBL) of 1,479 ± 831 ml. Twenty of 126 patients (15.9%) received 42 units of allogeneic packed red blood cells (PRBC), for a mean of 2.1 ± 1.2 U/patient. The transfusion rate was higher in patients with a preoperative hematocrit of 40% or less (45 vs. 13%, p = 0.014). Conclusions: Anatomical RRP is still associated with appreciable operative blood loss. Owing to the high preoperative hematocrit values, the allogeneic blood transfusion rate is low and the transfusion requirement of the majority of patients is limited to about 2 units of PRBC. Preoperative autologous blood augmentation strategies may not be routinely needed for patients with a basal hematocrit of >40%.

Hepatitis C Virus Infection An Endemic Area in North Italy

The prevalence of hepatotropic virus infections, as indicated by viral markers in the serum of blood donors, may vary considerably in different countries and in different areas within the same country [l]. A number of epidemiologic surveys [2,3] have shown that both hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are more frequent in southern than in northern regions of Italy. However, our data obtained from blood donors of Bergamo, a province of northern Italy, are in contrast with these conclusions. In 1981 we found that the prevalence of HBsAg and HBsAb was 8 and 57%, respectively. This high prevalence of HBsAg fell to 2.2% in 1992 when prophylactic measures and a specific vaccinal program against HBV infection had been undertaken. A high prevalence of HCV infection in blood donors was demonstrated by the results of a prospective study on posttransfusion hepatitis (PTH) conducted at Ospedali Riuniti di Bergamo. In 1985, when blood donors were not screened for serum alanine aminotransferase (ALT), 10 of 54 (18%) patients followed up during 6 months after transfusion developed non-A, non-B hepatitis. After introduction of systematic screeningfor ALT, the incidence of PTH decreased to 9.4% (10 of 106 patients). By the extensive use of first-generation Elisa test for HCV antibodies (anti-HCV) in 1990, the incidence of PTH was further reduced to 7.3% (5 of 68 patients). Finally, starting from February 1991, when the second-generation Elisa test for anti-HCV was available, none of 142 transfused patients developed non-A, non-B hepatitis. Using this test (Abbott, Chicago, Ill., USA) in 30,020 volunteer blood donors coming from Bergamo area, we found that 987 (3.27%) of them were anti-HCVpositive, this prevalence being one of the highest so far reported in the international literature [l]. In thiscontext, we believe that the large number of patients with HCV-associated cryoglobulinemia we have recently described [4] may reflect the high frequency of HCV infection in the general population of our area. It is also interesting to note that the territorial distribution of anti-HCV-positive blood donors was not homogeneous. In particular, we observed a greater than 5% prevalence in 24 villages and little towns placed in close proximity to the four rivers flowing in our province. The epidemiologic reasons for the high prevalence and the particular distribution of HCV carriers in our area are at present unknown. The common routes of transmission are well documented as being parenteral, such asvia blood transfusion and intravenous drug abuse [5], but these risk factors are not particularly high in our blood donor population. The possibility of waterborne enteral or percutaneous transmission is unlikely and seems to be excluded by preliminary results of our studies which failed to detect HCV RNA by polymerase chain reaction in samples of water. Other possibilities regarding inapparent parenteral transmission via more frequent interhuman contacts in places with higherdensityofpopulationorbitesofhematophagous arthropods swarming in these rivers, are the subject of current investigation. .............................................. References

Mixed Cryoglobulinaemia: A Guide to Drug Treatment

Mixed cryoglobulins are cold-precipitable serum proteins composed of at least 2 immunoglobulins. One of the components is a polyclonal IgG while the other, usually of the IgM class, may be polyclonal or monoclonal and acts as an anti-IgG rheumatoid factor. Mixed cryoglobulins are frequently detected in the serum of patients with infectious, immunological or lymphoproliferative disease; however, until recently, no identifiable disorder was found in 30 to 50% of all cryoglobulinaemic patients, their condition being defined as essential or idiopathic mixed cryoglobulinaemia. A large body of information is now accumulating in favour of the aetiological role of hepatitis C virus (HCV) in most of these patients. There is some evidence that HCV may enter lymphocytes and actively replicate in these cells. It is also conceivable that the presence of HCV in B lymphocytes may stimulate the production of anti-HCV antibodies which in turn, by binding together with HCV antigens and anti-IgG rheumatoid factors, form cryoprecipitable circulating immune complexes. This important progress in the understanding of mixed cryoglobulinaemia has revolutionised the treatment of this disease. In fact, interferon-α has been proposed as first choice therapy, its beneficial effect being attributed to the antiviral activity of the drug. However, interferon therapy, at least with the standard dosages currently used in chronic hepatitis C, is initially effective in 60% or fewer of cases and the long term response is generally disappointing. Moreover, because of the late appearance of the beneficial effect and the frequent occurrence of adverse drug reactions, interferon-α cannot be recommended for the acute flare-ups of disease. In the event of an exacerbation, traditional immunosuppressive therapy, which may be associated with plasma-exchange, still has considerable efficacy.