Serum oncostatin M in multiple myeloma: association with prognostic factors

We report on five children with haematological malignancies who underwent allogeneic peripheral blood progenitor cell (PBPC) transplantation. PBPC were harvested from HLA-identical sibling donors after G-CSF (10 μg/kg/d s.c.) mobilization. Aphereses were carried out on day 5 after G-CSF using a Cobe Spectra blood cell separator. All PBPC allografts were cryopreserved before transplantation. The median of CD34⁺ cells and CD3⁺ cells infused were 14.1×10⁶/kg recipient body weight (range 4.92–22.3) and 2.40×10⁸/kg recipient body weight (range 0.54–4.82), respectively. Engraftment occurred in all cases. The median time to a neutrophil count >0.5×10⁹/l and a platelet count >20×10⁹/l were 15 and 14 d, respectively. The incidence of severe acute graft-versus-host disease was 20%. These data suggest that allogeneic PBPC transplantation might be an alternative to bone marrow transplantation in children.     

Transplantation of HLA-mismatched CD34+ selected cells in patients with advanced malignancies: severe immunodeficiency and related complications

This trial was designed to test the use of CD34⁺ selected haemopoietic stem cells (HSC) in HLA-mismatched donor–recipient pairs, following intensive conditioning with thiotepa, antilymphocyte globulin (ALG), cyclophosphamide and single-dose total-body irradiation (sTBI). 10 patients aged 16–50 with advanced malignancies and a two- or three-antigen mismatched family donor entered this study. Donor marrow and G-CSF primed peripheral blood cells were processed separately on CD34 columns (Ceprate). The median number of infused CD34⁺ cells were 5.66 × 10⁶/kg, with 0.55 × 10⁶/kg CD3⁺ cells. Nine patients received cyclosporin for graft-versus-host disease (GvHD) prophylaxis. Median neutrophil counts on day 21 were 2 × 10⁹/l with a median platelet count of 60 × 10⁹/l, but CD4 counts remained extremely depressed throughout the study. Acute GvHD was scored as grade 0–I in two patients, as grade II in seven, and grade III in one. Eight patients died at a median interval of 72 d from HSCT (range 20–144) due to cytomegalovirus (CMV) associated interstitial pneumonitis (IP) ( n  = 5), renal failure ( n  = 1), GvHD ( n  = 1) and Aspergillus meningitis ( n  = 1). Two patients are alive 365–495 d post transplant, one in remission and one in relapse.
This study suggests that large numbers of positively selected mismatched HSC can rapidly engraft after intensive conditioning regimen: however, profound post-transplant immunodeficiency leads to a high risk of lethal infectious complications.     

Factors associated with successful mobilization of peripheral blood progenitor cells in 200 patients with lymphoid malignancies

Peripheral blood progenitor cells (PBPC) were mobilized and harvested in 200 patients treated for non-Hodgkin's lymphoma ( n  = 148), Hodgkin's disease ( n  = 22) and multiple myeloma ( n  = 30). The variables predicting the collection of a minimal (>2.5 × 10⁶/kg) or a high (>10 × 10⁶/kg) CD34⁺ cell count were analysed. Patients were mobilized with haemopoietic growth factors following either standard chemotherapy ( n  = 49) or high-dose cyclophosphamide, given alone ( n  = 55) or combined with high-dose VP16 ( n  = 86). 10 patients received haemopoietic growth factors only. The first mobilization resulted in a PBPC harvest with enough CD34⁺ cells in 179/200 patients (90%). High-dose cyclophosphamide, with or without VP16, did not mobilize a higher progenitor cell yield than standard chemotherapy. When performing multiple regression analysis in the 190 patients who received chemotherapy-containing mobilization, only the number of previous chemotherapy regimens and the exposure to fludarabine predicted for a failure to collect a minimal PBPC count ( P  = 0.06 and 0.0008 respectively). The target to collect a high CD34⁺ cell count was negatively associated with the number of previous chemotherapy regimens ( P  = 0.002). When only non-Hodgkin's lymphoma patients were considered for multivariate analysis, low-grade histology with fludarabine appeared to be associated with poor PBPC cell yield ( P  = 0.08 and 0.005 respectively). This data confirms that PBPC harvest should be planned early in the disease course in transplant candidates, and can be obtained after a standard course of chemotherapy.     

STEM CELL MOBILIzATION IN NORMAL DONORS FOR ALLOGENEIC TRANSPLANTATION: ANALYSIS OF SAFETY AND FACTORS AFFECTING EFFICACY

The use of peripheral blood stem cells instead of bone marrow as the source of haemopoietic cells for allogeneic transplantation is being increasingly explored. We have analysed data from 17 normal donors who underwent stem cell mobilization for allogeneic transplantation with an identical protocol using G-CSF at a dose of 10 μg/kg/d, with the first leukapheresis (LP) on the day following the fourth dose of G-CSF. Both G-CSF administration and leukapheresis were well tolerated. Donors underwent a median of two leukaphereses (range one to three) and a median of 6.80 × 10⁶ CD34⁺ cells/kg recipient weight (range 2.4–15.6 × 10⁶) were collected. The median number of CD34⁺ cells per kg donor weight was 6.05 × 10⁶, when corrected for a 12 litre leukapheresis, this gave a median total of 3.89 × 10⁶ CD34⁺ cells/kg donor weight. When analysed with respect to factors which might influence the efficacy of mobilization, male donors were associated with a superior yield. The median number of CD34⁺ cells/kg/LP harvested was 4.96 × 10⁶ in males and 2.79 × 10⁶ in females ( P <0.05). The results suggested that, given a recipient of 75 kg, in a male donor a single 12 litre leukapheresis should yield sufficient CD34⁺ cells (4 × 10⁶/kg), whereas a female donor would be likely to need two leukaphereses. Age was not found to affect donor yield. In summary, these data confirm that leukapheresis is a safe procedure in normal donors and suggest that males may be more efficient mobilizers of stem cells than females.     

Collection of peripheral blood stem cells from normal donors 60 years of age or older

We report 14 normal peripheral blood stem cell (PBSC) donors ≥ 60 years of age who had cytokine mobilization followed by PBSC apheresis for allogeneic transplantation. Mobilization was achieved with filgrastim (6 μg/kg twice daily). Their median age was 63.5 years (range 60–77), and 43% had a positive medical history, mainly hypertension and/or cardiac problems. Their median pre-apheresis leucocyte count (×10⁹/l) was 38.6 (range 29.6–63.4). The median apheresis yield (×10⁶ CD34⁺ cells/litre blood processed, first apheresis) was 27.9 (range 1.6–54.8). The target cell dose (≥4& times; 10⁶ CD34⁺ cells/kg recipient) was reached with one procedure in eight (57%) donors. Filgrastim-related adverse events were acceptable and apheresis was well tolerated. When compared to younger donors (<60 years of age), a trend to a lower CD34⁺ apheresis yield and a requirement for more than one apheresis to achieve the collection target (≥4 ×10⁶ CD34⁺ cells/kg) was evident. Although older (≥60 years) donors seem to mobilize less effectively, these data suggest that PBSC collection from them is feasible and has an acceptable short-term safety profile.     

Immunomagnetic selection of CD34+peripheral blood stem cells for autografting in patients with breast cancer

Contamination of transplants with tumour cells may contribute to relapse after peripheral blood stem cell transplantation (PBSCT). We studied the feasibility of CD34⁺ cell selection from blood-derived autografts obtained following G-CSF-supported cytotoxic chemotherapy in a group of 25 patients with breast cancer (10 with high-risk stage II/III and 15 with stage IV without bone or bone marrow involvement).
Using immunomagnetic beads (Isolex 300 SA, Baxter) CD34⁺ cells were enriched and released by chymopapain resulting in a median purity of 95% (range 82–99%) and a median recovery of 80% (range 27–132%). The enrichment procedure did not change the proportion of CD34⁺ subsets coexpressing HLA-DR, CD38 and Thy-1, while L-selectin was removed from the cell surface following selection. Using a sensitive immunocytological technique with a cocktail of epithelial-specific antibodies (anti-cytokeratin 8, 18 and 19; HEA125; BM7 and BM8), five leukaphereses products contained epithelial cells, whereas the selected CD34⁺ cell fraction was free of tumour cells. A neutrophil count of 0.5×10⁹/l and a platelet count of 20×10⁹/l was reached after a median time of 14 and 10 d following 40 high-dose chemotherapy (HDC) cycles. Our results indicate that immunomagnetic selection of CD34⁺ cells yields highly purified autografts devoid of tumour cells whereas the engraftment ability of the progenitor and stem cells is fully retained.     

Feasibility of peripheral blood progenitor cell harvest and transplantation in patients with poor-risk myelodysplastic syndromes

Myelodysplastic syndromes (MDS) are a group of clonal haematological disorders with a highly unfavourable prognosis. Allogeneic bone marrow transplantation offers the sole possibility for cure and prolonged survival, but is only available for a minority of patients. Therefore, we investigated the feasibility of PBPC collection and transplantation in 11 patients with high-risk myelodysplasia who were not eligible for allogeneic bone marrow transplantation. In six patients, PBPC were harvested after mobilization with G-CSF alone. Five patients were harvested during the recovery phase of intensive chemotherapy combined with G-CSF. This resulted in seven patients in an adequate CD34 progenitor yield >1 × 10⁶/kg. Six patients obtained a CFU-GM content of the PBPC harvest >10 ×10⁴/kg. Five patients were subsequently transplanted following a standard BuCy4 regimen. The median to ANC (absolute neutrophil count) ≥0.5 and 1.0 × 10⁹/l was respectively 14 d (range 10–18) and 16 d (range 11–25). Platelets were self-supporting ≥20 × 10⁹/l after a median of 41 d (range 8–144). One patient had a persistent lack of platelet engraftment unresponsive to infusion of back-up bone marrow.
These data demonstrate that in selected patients with high-risk MDS, adequate PBPC collection appears feasible, enabling the harvest of sufficient cell numbers required for rapid and stable engraftment after reinfusion. Improvement in mobilization efficiency may enable the collection of higher CD34⁺ progenitor cell numbers required for more rapid platelet engraftment. PBPC transplantation may be an alternative treatment option for patients who lack an allogeneic marrow donor. Follow-up is, however, still too limited to draw any conclusion regarding the long-term cure rate.     

COLLECTION AND TRANSPLANTATION OF PERIPHERAL BLOOD PROGENITOR CELLS MOBILIzED BY G-CSF ALONE IN CHILDREN WITH MALIGNANCIES

Results of collection and transplantation of peripheral blood progenitor cells (PBPC) mobilized by G-CSF in 31 children with different malignancies were analysed. A total of 43 aphereses were performed, following administration of granulocyte colony-stimulating factor (G-CSF), using a continuous flow blood cell separator (Cobe Spectra) through a central venous catheter. For patients weighing ≤25 kg the extracorporeal line was primed with red blood cells. The mean blood flow rate was 33.2ml/min (range 12–56ml/min). The mean number of mononuclear cells (MNC), granulocyte-macrophage colony-forming units (CFU-GM) and CD34⁺ cells collected were 7.22×10⁸/kg body weight (b.w.), 15.9×10⁴/kg and 5.44×10⁶/kg respectively.
The morbidity related to PBPC collection was low and the mean apheresis time was 302.7min (range 115–492). The volume of processed blood for apheresis (per kilogram body weight) ranged from 117 to 539.6 (mean 309.13ml/kg). 20 patients required only one apheresis to collect the minimum requirement of 5–7×10⁸/kg of MNC.
All patients subsequently underwent autografting with PBPC after myeloablative therapy. Days to achieve an absolute count of neutrophils (ANC) <0.5×10⁹/l and a platelet count of 20×10⁹2/l without platelet support were 9.5 and 18, respectively. The number of CD34⁺ cells infused correlated highly with engraftment kinetics. The extramedullary toxicity was low and manageable.     

Increased number of peripheral blood CD34+ cells in lithium-treated patients

Eight adult patients with bipolar disorder were prospectively examined to find whether lithium carbonate increased their peripheral blood CD34⁺ haemopoietic stem cells. Following lithium therapy for 3–4 weeks their neutrophil counts increased by a mean of 88% (from 4625 ± 1350 × 10⁹/l, mean ± SD pretreatment, to a peak of 8300 ± 3910 × 10⁹/l). Concommitantly, there was a significant increment in their CD34⁺ cells (from 0.11 ± 0.01% to a peak of 0.18 ± 0.08%). There was a significant correlation between the rise in neutrophil count and that of the CD34⁺ cells ( r  = 0.795, P  = 0.019). Lithium therapy may be used to mobilize peripheral blood CD34⁺ cells for marrow transplantation.     

Factors influencing collection of peripheral blood progenitor cells following high-dose cyclophosphamide and granulocyte colony-stimulating factor in patients with multiple myeloma

We treated 103 multiple myeloma (MM) patients with 7 g/m² cyclophosphamide (Cy) followed by 300 μg G-CSF/d to harvest peripheral blood progenitor cells (PBPC). PBPC autografts containing > 2.0 × 10⁶ CD34⁺ cells per kg body weight were obtained at the first attempt from 90/100 evaluable patients. The most significant factor predicting impairment of PBPC collection was the duration of previous melphalan treatment ( P  < 0.0001). In multivariate discriminate analysis, treatment with melphalan during the most recent chemotherapy cycles prior to mobilization ( P  = 0.0727) and previous radiotherapy ( P  = 0.0628) had a marginally significant negative influence on the efficacy of PBPC collection. We found no reduced functional capacity of CD34⁺ cells to restore haemopoiesis after myeloablative treatment related to the duration of melphalan exposure. At the time of best response to conventional treatment, a median paraprotein reduction of 21% was achieved following high-dose cyclophosphamide (HD-Cy). Two heavily pretreated patients died and one patient developed pulmonary toxicity W.H.O. grade IV following HD-Cy. Potential transplant candidates should undergo mobilization and harvesting of PBPC before melphalan-containing treatment. Combinations of haemopoietic growth factors and their dose modifications should be investigated to improve PBPC collection, to allow a dosage reduction of the mobilization chemotherapy.     

Granulocyte colony-stimulating factor given in addition to interferon-α to mobilize peripheral blood stem cells for autologous transplantation in chronic myeloid leukaemia

In order to potentially mobilize and harvest the Ph⁻ cells observed in most patients with chronic myeloid leukaemia (CML) during interferon-α (IF-α) therapy, G-CSF (filgrastim), 5 μg/kg/d, was administered subcutaneously together with IF-α to 30 CML patients in haematological remission but with various degrees of cytogenetic remission, after IF-α therapy. Peripheral blood stem cells (PBSC ) were harvested using standard aphereses from day 5 of G-CSF. Patients underwent one to four (median three) aphereses. Median total yields/kg were 7.6 (range 3.8–25) × 10⁸ MNC, 3.4 (0–140) × 10⁶ CD34⁺ cells, and 17 (1.1–107) × 10⁴ CFU-GM. No patient had a significant increase in the percentage of Ph⁺ cells in the bone marrow under G-CSF therapy. The percentage of Ph⁺ cells in apheresis products tended to decrease between the first and the last apheresis ( P  = 0.05). 14 patients who were not responsive to IF-α were transplanted after conditioning with busulphan 16 mg/kg and melphalan 140 mg/m². Median time to neutrophils > 0.5 × 10⁹/l was 20 d (16–114 d) and to platelets > 50 × 10⁹/l 18 d (12–149 d). Nine patients had a major cytogenetic response post graft, which correlated with the amount of Ph⁺ cells reinfused with the graft ( P  = 0.02). We conclude that this procedure is feasible, allowing the harvest of enough PBSC, some of them Ph⁻ in patients who responded to IF-α, to allow autologous transplantation.     

Progenitor cell subsets and engraftment kinetics in children undergoing autologous peripheral blood stem cell transplantation

The main objective of the present study was to determine the role of CD34⁺ cell subsets in the haemopoietic recovery of children undergoing peripheral blood stem cell transplantation. For this purpose, 38 leukaphereses from 33 children with malignancies mobilized with G-CSF were analysed. Using dual-colour flow cytometry, different subpopulations of CD34⁺ cells were quantified and the number of each reinfused subsets correlated with haemopoietic resurgence. Multivariate analysis showed that the number of CD34⁺CD38⁻ cells and CD34⁺CD38⁺ cells correlated better with time to neutrophil and platelet recovery, respectively, than the total number of CD34⁺ cells. Threshold values for rapid haemopoietic recovery, determined by the receiver operating characteristic analysis, were found to be 0.5 × 10⁶ CD34⁺CD38⁻ cells for neutrophil engraftment, and 2.0 × 10⁶ CD34⁺CD38⁺ cells for platelet recovery. It is suggested that the analysis of CD34⁺ cell subsets could increase understanding of the repopulation capacity of a given leukapheresis product in peripheral blood stem cell transplantation procedures in children. In particular, this procedure could be extremely useful when low numbers of CD34⁺ cells are collected.     

Mobilization and transplantation of Philadelphia chromosome-negative peripheral blood progenitor cells in patients with CML

Mobilization and transplantation of peripheral blood progenitor cells (PBPCs) was investigated in patients with stage IA or stage IIA chronic myelogenous leukaemia (CML) using combination chemotherapy with idarubicin, cytosine arabinoside and etoposide (ICE) followed by simultaneous administration of rhG-CSF and rhIL-2 or rhG-CSF alone. 17 patients (stage IA: 12; stage IIA: five) were mobilized. Chemotherapy, cytokine priming and collection of PBPCs were well tolerated. Using large-volume aphereses, a median number of 10.6 × 10⁷/kg b.w. of MNC were harvested, which yielded between 0.15 and 21.0 × 10⁶ CD34⁺ cells/kg b.w. High numbers of CD34⁺ HLA-DR⁻ cells could be obtained. Autologous transplantation of mostly Ph⁻ apheresis products was performed in 16/17 patients after high-dose chemotherapy. 10 patients achieved a complete or major cytogenetic remission (stage IA: seven; stage IIA: three), but six patients did not respond cytogenetically. Median follow-up after transplantation was 18+ months. Our data show that this very efficient treatment modality for PBPC mobilization in CML was safe and able to induce major and sustained cytogenetic responses in the majority of patients.     

Endothelial cell precursors are normal components of human umbilical cord blood

Endothelial cells are part of the normal bone marrow stroma. We have previously shown human umbilical cord blood (UCB) does not produce stroma in standard long-term cultures. Highly enriched (93–98%) UCB CD34⁺ cells were cultured for 6 weeks with interleukin-2 and conditioned medium from the 5637 carcinoma cell line ( n  = 4). The resulting 'fibroblast like' cells were shown to be endothelial by expression of von Willebrand factor (VWF), ICAM-1 (CD54), E-selectin (CD62^E) and PECAM (CD31). Endothelial monolayers seeded with CD34⁺ UCB cells supported expansion of colony forming cells and CD34⁺ cells. We conclude that endothelial cell precursors circulate in UCB, and may be derived from the CD34⁺ cell fraction.     

Flow cytometric detection of cytomegalovirus antigen in peripheral blood cells after bone marrow transplantation

We report 13 normal peripheral blood stem cell (PBSC) donors who had a second PBSC collection for allogeneic transplantation performed after the first. The median interval between the first and second collection was 5 months. Mobilization was achieved with filgrastim (12 μg/kg/d). No significant difference was found in the median pre-apheresis leucocyte count (×10⁹/l) between the two donations (40.2 v 38.5; P  = 0.91). The median apheresis yield (×10⁶ CD34⁺ cells/litre blood processed, first apheresis) was also similar (28 v 27.3; P  = 0.91). Filgrastim-related adverse events were comparable. These data suggest that second PBSC collections are feasible, similarly tolerated and provide comparable apheresis yields.     

Sustained decrease of peripheral lymphocytes after allogeneic blood stem cell aphereses

48 healthy donors underwent peripheral blood stem cell (PBSC) apheresis for allogeneic transplantation beginning on day 4 of G-CSF (2 × 5 μg/kg) mobilization. In one to four (median two) large-volume mononuclear cell aphereses, a median of 55.9 × 10⁹ of lymphocytes (range 21.0–109.2 × 10⁹) were collected, an amount comparable to lymphocyte numbers removed by therapeutic lymphaphereses in autoimmune diseases. Mean peripheral lymphocyte counts decreased from premobilization values of 2.31 × 10⁹/l to 1.31 × 10⁹/l at a median of 34 d (1 month) and 1.53 × 10⁹/l at a median of 327 d (11 months). The decrease in peripheral lymphocyte counts was significantly correlated with the number of lymphocytes removed and the number of aphereses. Neutrophil and platelet counts returned to normal values after 1 month whereas monocyte counts and haemoglobin concentrations were significantly decreased at 1 month but not at 11 months.     

Combined negative and positive selection of mobilized CD34+ blood cells

We tested four negative and two positive selection methods for separation of CD34⁺ cells from mobilized blood cells, and analysed fold-enrichment, purity and recovery of CD34⁺ cells after selection procedures. The elimination of mature CD34⁻ cells was achieved by adhesion to nylon-wool fibre (5.9 ± 1.0 mean fold-enrichment and 65.2 ± 2.3 mean recovery of CD34⁺ cells). Standard or modified Ficoll-Hypaque and Percoll density gradients, as well as phagocytosis with magnetic beads, were less effective in eliminating CD34⁻ cells, both purity and fold-enrichment of CD34⁺ cells being lower than those obtained with separation by nylon-wool. Both positive selection methods tested, Ceprate and MiniMacs System, generated highly purified CD34⁺ cell populations ranging from 80% to 90%. The recovery of CD34⁺ cells was optimal with MiniMacs (77.9±3.6) and low with Ceprate (28.8±2.8). Based on these results, in two large-scale experiments we combined nylon-wool fibre and MiniMacs System in a two-step separation procedure obtaining a 36.9±2.6 mean fold-enrichment and a 50.5±0.3 mean recovery of CD34⁺ cells. In this way we achieved optimal enrichment and recovery of CD34⁺ cells, with a substantial saving of cost compared to either selection method alone.     

Influence of bone marrow graft B lymphocyte subsets on outcome after HLA-identical sibling transplants

The potential role of the infused B cell subset after Hematopoietic Stem Cell Transplantation has not been yet studied. The present study analyzed the impact of B cells on transplant outcome in 254 patients who received a bone marrow graft from a human leucocyte antigen-identical sibling donor. The influence of B lineage-specific hematopoietic progenitor cells (CD34⁺ CD19⁺) and B cells (immature and mature B cells, CD34⁻ CD19⁺) was also analyzed. All included patients received a myeloablative regimen. The cumulative incidence function of acute graft-versus-host (GvHD) grade II to IV was 48% and was inversely associated with the number of CD34⁺ CD19⁺. There were no statistically significant associations between B cell subsets and chronic GvHD or survival. The CD34⁺ CD19⁺ B cell subset remained significantly associated with acute GvHD in multivariate analysis (Relative risk = 0·32, 95% confidence interval: 0·11–0·92, P  =   0·035). In conclusion, a higher B lineage-specific hematopoietic progenitor cells (CD34⁺ CD19⁺) cell dose is associated with a significant decrease incidence of acute GvHD.     

Second transplantation with CD34+ bone marrow cells selected from a two-loci HLA-mismatched sibling for a patient with chronic myeloid leukaemia

A 43-year-old man with chronic myeloid leukaemia underwent a second transplant with CD34⁺ bone marrow cells selected from his two-loci HLA-mismatched sibling after rejection of the first graft from an HLA-matched unrelated donor. By immunomagnetic positive selection, CD34⁺ marrow cells at 0.95×10⁶/kg with 97% purity and CD3⁺ T lymphocytes at 1.3×10⁴/kg were collected and transplanted. Engraftment was confirmed to be of CD34⁺ cell-donor origin. The patient developed only grade I acute graft-versus-host disease (GVHD) and no chronic GVHD to date. These observations suggest that allogeneic CD34⁺ bone marrow cells are capable of reconstituting haemopoiesis and that CD34⁺ selection could be applicable to T-cell depletion.     

Hepatitis G virus infection in patients with bleeding disorders

Eighty-two patients with bleeding disorders registered with our centre were screened for infection with hepatitis G virus (HGV). 80 patients were positive for hepatitis C (HCV) antibodies, 66 of whom (83%) were HCV PCR positive. 11 patients (13%) were HGV RNA-positive, a similar prevalence rate to that of other studies of patients with bleeding disorders who received factor concentrates prior to the introduction of viral inactivation procedures. There was no significant difference in histological activity index (HAI) between the 10 HGV RNA-positive and the 31 HGV RNA-negative patients who underwent liver biopsy for assessment of HCV infection (median HAI scores 5.5, range 2–10 and four, range 0–10 respectively, P  = 0.07). One patient in each group had established cirrhosis. In patients who underwent HCV quantitation there was no significant difference in HCV viral titre between HGV RNA-positive and negative patients (median HCV titre in HGV RNA-positive patients 2.10 × 10⁵ DNA copies /ml ( n  = 8) range 4.17 × 10² to 4.17 × 10⁶, median HCV titre in HGV RNA-negative patients 3.33 × 10⁵ ( n  = 31) range 1.00 × 10³ to 6.67 × 10⁶, P  = 0.68). In this study there was no evidence that individuals co-infected with HGV and HCV have more severe liver disease than those infected with HCV alone.