Unaltered lymphocyte subsets in hepatitis C virus-seropositive blood donors H.E. PRINCEAND C.T. FANG Lymphocyte subsets were evaluated by dual-color flow cytometty in whole blood specimens from 35 blood donors who were seropositive on enzyme-linked immunosorbent assay (ELISA) for hepatitis C virus (HCV) and whose sera reacted in a four-antigen recombinant immunoblot assa (RIBA) referred to as the HCV+ R group), 15 donors who were seropositive on ELI A for H V with indeterminate or negative RIBA results (the HCV+ I/N group), and 25 HCV-seronegative controls (HCV- group). The cell subsets assessed included natural killer cells, B cells, T cells, CD4 and CD8 subsets of T cells, and T-cell subsets defined by the coexpression of markers that appear (HLA-DR, CD25, CD38) or disappear (CD45RA) after activation. A one-way analysis of variance revealed no significant differences among the three study groups. These findings show that, unlike cytomegalovirus- and human immunodeficiency virus-positive individuals, HCV-positive individuals d o not exhibit lymphocyte alterations indicative of the immune activation caused by chronic viral infection. TRANSFUSION 1992;32:166-168.
g
A
Abbreviations: ALT = aianlne amlnotransferase; anti-HBc = antlbody to hepatitis B core antigen; ELSA = enzyme-ilnked lmmunosorbent assay; HCV = hepatitls C virus; HCV= HCV-ELSA and RIBA-negatlve group; HCV+I/N = HCV ELISA-positive and RIBAIndeterminate or -negative group; HCV+ R = HCV-EUSA-positive and RIBA-positive group; RIBA = four-antigen recomblnant fmrnunoblot assay.
HEPATITISC VIRUS (HCV) is a recently discovered RNA virus associated with transfusion-transmitted nonA,non-B hepatitis.' An enzyme-linked immunosorbent assay (ELISA) to detect antibodies recognizing the C100 antigen of HCV is now used for screening all donated blood components. A four-antigen recombinant immunoblot assay (RIBA), incorporating two forms of the C-100 antigen plus two additional HCV antigens, was recently described as a possible confirmatory assay for infective HCV-ELISA-positive blood donors.2
Materials and Methods Cell preparation and staining We obtained the centrifuged EDTA tube of blood used for red cell typing of 50 blood donors with HCV-ELISA absorbance values >2.5. Tubes from 25 HCV-negative donors were randomly selected from the same racks (originating from the same mobile collection site) as were those from the HCVpositive donors. The interval between specimen collection at the mobile site and the analyses described here was approximately 24 hours. The HCV-positive donors studied were apparently asymptomatic, and no assessments of hepatic histology were performed; the length of time since infection is unknown. After saving 0.5 mL of plasma for RIBA testing (see below), we added 0.5 mL of phosphate-buffered saline to each tube, recapped the tube, and mixed the contents on a mechanical rocker for 10 minutes. Blood was then distributed among several 12 x 75 mm plastic tubes (0.05 mL per tube), each containing a pair of monoclonal antibodies (Becton Dickinson Immunocytometry Systems, San Jose, CA) shown to define specific subsets of lymphocytes. The CD markers recognized by the monoclonal antibody pairs and the characteristics of the cells are included in the tables in the Results section. One antibody of each pair was tagged with phycoerythrin, which emits red fluorescence when excited by a 488 nm laser, and the other antibody was tagged with fluorescein, which emits green fluorescence. Staining controls included phycoerythdnand fluorescein-tagged nonspecific subclass controls and single-stain phycoerythrin-anti-CD3 and fluorescein-anti-CD3 controls for adjusting compensation. We incubated cells with monoclonal antibodies for 30 minutes at 4°C and then added O.lSM(0.15 molk) buffered ammonium chloride to each tube. After 10 minutes at room temperature, we centrifuged the tubes,
Polymerase chain reaction studies2 have shown that some HCV-positive persons still carry detectable viral genome several years after seroconversion, which indicates chronic infection. Because chronic viral infections may be associated with immune activation due to chronic antigenic s t i r n u l a t i ~ n , we ~ . ~ sought to determine if HCVpositive blood donors showed evidence of immune system activation. In addition to measuring the major lymphocyte subsets, we assessed the expression of cell surface markers that were suggestive of in vivo activation. From the American Red Cross Blood Services, Los Angeles/Orange Counties Region, Los Angeles, California, and Jerome H. Holland Laboratory, American Rcd Cross, Rockville, Maryland. Supported in part by funds from the Biomedical Research and Development Program of American Rcd Cross Blood Services National Hcadquarters. Rcccivcd for publication June 5 , 1991; revision received July 23, 1991, and accepted July 24, 1991.
166
TRANSFUSION 1992-Vol. 32. No. 2
167
LYMPHOCYTE SUBSETS IN HCV INFECTION
Table 1. Lymphocyte subsets* in hepatitis C virus (HCV) infection HCV reactivity Cluster measured HCV - t HCV + I/N* HCV + R§ CD19 B cells 11 2 6 14 f 5 14 f 6 Natural killer cells CD16 + CD56 17 f 5 15 f 6 14 f 5 T cells CD3 76 f 7 75 f 6 76 f 8 Helper/lnducer T cells CD4 51 f 6 50 f 8 49 f 7 Suppressorlcylotoxic T cells CD8 31 9 29 f 8 32 f 8 ' Data represent the mean percentage of positive lymphocytes f 1 SD. t HCV-enzyme-linked immunosorbent assay (ELSA)- and four-antigen recombinant immunoblot assay (RIBA)-negativecontrols. Donors who were HCV-positive and RIBA indeterminant or negative. 8 HCV-ELISA positive donors who were positive on RIBA.
Cell subset
*
+
Table 2. Activated T-cell subsets* in hepatitis C virus HCV infection Subset
Second marker
CD4 CD4 CD4 CD4 CD8 CD8 CD8 CD8 CD8
CD45RA HLA-DR CD38 CD25 CD45RA
Dual characteristics of positive cells
HCV reactivity HCV-t
HCV + IINS
55 f 12 55* 9 Naive Activated l l f 5 11f 4 57 f 11 Actlvated/lmmature 59 t 12 Activatedlmemory 35 f 10 332 8 Naive 822 9 812 9 HIA-DR Activated 24 f 14 202 6 CD38 Activated/immature 60 f 13 54 f 12 CD25 Activated/memory 7 2 3 8f 3 CD57 Non-major histocompatibility 34 f 14 28 f 14 complex-restricted cytotoxicity * Data represent the mean proportion (%) of the indicated subset expressing the second marker indicated f 1 SD. t HCV-enzyme-linked immunoassay (ELSA)- and four-antigen recombinant immunoblot assay (RIBA)-negativecontrols. $ HCV-ELISA-positive donors with indeterminate or negative results on RIBA. § HCV-ELISA-positive donors with positive results on RIBA.
aspirated the supernatant, and resuspended the cells in 0.5 mL of phosphate-buffered saline containing 1 percent newborn calf serum. Because the packed cell and plasma volumes of the blood samples had been perturbed by prior red cell typing, accurate absolute white cell and lymphocyte counts could not be determined.
Flow cytometric analysis We analyzed the cells with a flow cytometer (FACScan, Becton Dickinson), using correlated analysis of forward- and right-angle (90") scatter to establish a lymphocyte gate. Fluorescence detectors were then calibrated with nonspecific binding controls; we adjusted the compensation by using the cells stained with phycoerythrin-anti-CD3 and fluorescein-anti-CD3. List mode data for 10,000 ungated events were collected and stored for each of the tubes. We subsequently analyzed the fluorescence of cells within the lymphocyte gate with research software (FACScan, Becton Dickins~n).~
Four-antigen RIBA We thawed cryopreserved plasma samples from the 50 HCVELISA-positive and 25 HCV-ELISA-negative donors and analyzed them for HCV reactivity by using a four-antigen RIBA (Chiron, Emeryville, CA). Interpretations were made following the suggestions of the manufacturer.
Results Of 50 HCV-ELISA-positive specimens, 35 were positive on four-antigen RIBA (HCV + R group); 4 HCV-ELISA-positive
HCV + R§ 49 f 12 12 2 6 54 f 11 382 9 80k 9 27 2 14
59 f 11 102 5 28 f 14
specimens were indeterminate, and 11were negative (HCV + I/ N group). All of the 25 HCV-ELISA-negative specimens were RIBA negative (HCV - group). The three study groups (HCV + R, HCV-I/N, HCV - ) did not differ significantly in a one-way analysis of variance with regard to age or gender (significance defined as ~ ~ 0 . 0 5 Only ). 1 of 25 HCV- persons had an elevated alanine transaminase (ALT) value, and none was positive for antibody to hepatitis B core antigen (anti-HBc); 1of the 15 HCV + I/N persons had an elevated ALT, and 3 were positive for anti-HBc. In the HCV + R group, 14 of 35 persons had ALT elevations only, 6 were positive for anti-HBc only, and 4 had both. Values for the major lymphocyte subsets are shown in Table 1. In a one-way analysis of variance, no significant differences were observed (significance defined as p < 0.05). Additional monoclonal antibody pairs measured T-cell subsets that were indicative of in vivo activation. As shown in Table 2, we observed no significant differences. There were no significant differences in any parameter measured when we compared HCV + R individuals with ALT elevations and/or anti-HBc and HCV + R individuals without these alterations (Mann-Whitney U test, significance defined as p
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A
Abbreviations: ALT = aianlne amlnotransferase; anti-HBc = antlbody to hepatitis B core antigen; ELSA = enzyme-ilnked lmmunosorbent assay; HCV = hepatitls C virus; HCV= HCV-ELSA and RIBA-negatlve group; HCV+I/N = HCV ELISA-positive and RIBAIndeterminate or -negative group; HCV+ R = HCV-EUSA-positive and RIBA-positive group; RIBA = four-antigen recomblnant fmrnunoblot assay.
HEPATITISC VIRUS (HCV) is a recently discovered RNA virus associated with transfusion-transmitted nonA,non-B hepatitis.' An enzyme-linked immunosorbent assay (ELISA) to detect antibodies recognizing the C100 antigen of HCV is now used for screening all donated blood components. A four-antigen recombinant immunoblot assay (RIBA), incorporating two forms of the C-100 antigen plus two additional HCV antigens, was recently described as a possible confirmatory assay for infective HCV-ELISA-positive blood donors.2
Materials and Methods Cell preparation and staining We obtained the centrifuged EDTA tube of blood used for red cell typing of 50 blood donors with HCV-ELISA absorbance values >2.5. Tubes from 25 HCV-negative donors were randomly selected from the same racks (originating from the same mobile collection site) as were those from the HCVpositive donors. The interval between specimen collection at the mobile site and the analyses described here was approximately 24 hours. The HCV-positive donors studied were apparently asymptomatic, and no assessments of hepatic histology were performed; the length of time since infection is unknown. After saving 0.5 mL of plasma for RIBA testing (see below), we added 0.5 mL of phosphate-buffered saline to each tube, recapped the tube, and mixed the contents on a mechanical rocker for 10 minutes. Blood was then distributed among several 12 x 75 mm plastic tubes (0.05 mL per tube), each containing a pair of monoclonal antibodies (Becton Dickinson Immunocytometry Systems, San Jose, CA) shown to define specific subsets of lymphocytes. The CD markers recognized by the monoclonal antibody pairs and the characteristics of the cells are included in the tables in the Results section. One antibody of each pair was tagged with phycoerythrin, which emits red fluorescence when excited by a 488 nm laser, and the other antibody was tagged with fluorescein, which emits green fluorescence. Staining controls included phycoerythdnand fluorescein-tagged nonspecific subclass controls and single-stain phycoerythrin-anti-CD3 and fluorescein-anti-CD3 controls for adjusting compensation. We incubated cells with monoclonal antibodies for 30 minutes at 4°C and then added O.lSM(0.15 molk) buffered ammonium chloride to each tube. After 10 minutes at room temperature, we centrifuged the tubes,
Polymerase chain reaction studies2 have shown that some HCV-positive persons still carry detectable viral genome several years after seroconversion, which indicates chronic infection. Because chronic viral infections may be associated with immune activation due to chronic antigenic s t i r n u l a t i ~ n , we ~ . ~ sought to determine if HCVpositive blood donors showed evidence of immune system activation. In addition to measuring the major lymphocyte subsets, we assessed the expression of cell surface markers that were suggestive of in vivo activation. From the American Red Cross Blood Services, Los Angeles/Orange Counties Region, Los Angeles, California, and Jerome H. Holland Laboratory, American Rcd Cross, Rockville, Maryland. Supported in part by funds from the Biomedical Research and Development Program of American Rcd Cross Blood Services National Hcadquarters. Rcccivcd for publication June 5 , 1991; revision received July 23, 1991, and accepted July 24, 1991.
166
TRANSFUSION 1992-Vol. 32. No. 2
167
LYMPHOCYTE SUBSETS IN HCV INFECTION
Table 1. Lymphocyte subsets* in hepatitis C virus (HCV) infection HCV reactivity Cluster measured HCV - t HCV + I/N* HCV + R§ CD19 B cells 11 2 6 14 f 5 14 f 6 Natural killer cells CD16 + CD56 17 f 5 15 f 6 14 f 5 T cells CD3 76 f 7 75 f 6 76 f 8 Helper/lnducer T cells CD4 51 f 6 50 f 8 49 f 7 Suppressorlcylotoxic T cells CD8 31 9 29 f 8 32 f 8 ' Data represent the mean percentage of positive lymphocytes f 1 SD. t HCV-enzyme-linked immunosorbent assay (ELSA)- and four-antigen recombinant immunoblot assay (RIBA)-negativecontrols. Donors who were HCV-positive and RIBA indeterminant or negative. 8 HCV-ELISA positive donors who were positive on RIBA.
Cell subset
*
+
Table 2. Activated T-cell subsets* in hepatitis C virus HCV infection Subset
Second marker
CD4 CD4 CD4 CD4 CD8 CD8 CD8 CD8 CD8
CD45RA HLA-DR CD38 CD25 CD45RA
Dual characteristics of positive cells
HCV reactivity HCV-t
HCV + IINS
55 f 12 55* 9 Naive Activated l l f 5 11f 4 57 f 11 Actlvated/lmmature 59 t 12 Activatedlmemory 35 f 10 332 8 Naive 822 9 812 9 HIA-DR Activated 24 f 14 202 6 CD38 Activated/immature 60 f 13 54 f 12 CD25 Activated/memory 7 2 3 8f 3 CD57 Non-major histocompatibility 34 f 14 28 f 14 complex-restricted cytotoxicity * Data represent the mean proportion (%) of the indicated subset expressing the second marker indicated f 1 SD. t HCV-enzyme-linked immunoassay (ELSA)- and four-antigen recombinant immunoblot assay (RIBA)-negativecontrols. $ HCV-ELISA-positive donors with indeterminate or negative results on RIBA. § HCV-ELISA-positive donors with positive results on RIBA.
aspirated the supernatant, and resuspended the cells in 0.5 mL of phosphate-buffered saline containing 1 percent newborn calf serum. Because the packed cell and plasma volumes of the blood samples had been perturbed by prior red cell typing, accurate absolute white cell and lymphocyte counts could not be determined.
Flow cytometric analysis We analyzed the cells with a flow cytometer (FACScan, Becton Dickinson), using correlated analysis of forward- and right-angle (90") scatter to establish a lymphocyte gate. Fluorescence detectors were then calibrated with nonspecific binding controls; we adjusted the compensation by using the cells stained with phycoerythrin-anti-CD3 and fluorescein-anti-CD3. List mode data for 10,000 ungated events were collected and stored for each of the tubes. We subsequently analyzed the fluorescence of cells within the lymphocyte gate with research software (FACScan, Becton Dickins~n).~
Four-antigen RIBA We thawed cryopreserved plasma samples from the 50 HCVELISA-positive and 25 HCV-ELISA-negative donors and analyzed them for HCV reactivity by using a four-antigen RIBA (Chiron, Emeryville, CA). Interpretations were made following the suggestions of the manufacturer.
Results Of 50 HCV-ELISA-positive specimens, 35 were positive on four-antigen RIBA (HCV + R group); 4 HCV-ELISA-positive
HCV + R§ 49 f 12 12 2 6 54 f 11 382 9 80k 9 27 2 14
59 f 11 102 5 28 f 14
specimens were indeterminate, and 11were negative (HCV + I/ N group). All of the 25 HCV-ELISA-negative specimens were RIBA negative (HCV - group). The three study groups (HCV + R, HCV-I/N, HCV - ) did not differ significantly in a one-way analysis of variance with regard to age or gender (significance defined as ~ ~ 0 . 0 5 Only ). 1 of 25 HCV- persons had an elevated alanine transaminase (ALT) value, and none was positive for antibody to hepatitis B core antigen (anti-HBc); 1of the 15 HCV + I/N persons had an elevated ALT, and 3 were positive for anti-HBc. In the HCV + R group, 14 of 35 persons had ALT elevations only, 6 were positive for anti-HBc only, and 4 had both. Values for the major lymphocyte subsets are shown in Table 1. In a one-way analysis of variance, no significant differences were observed (significance defined as p < 0.05). Additional monoclonal antibody pairs measured T-cell subsets that were indicative of in vivo activation. As shown in Table 2, we observed no significant differences. There were no significant differences in any parameter measured when we compared HCV + R individuals with ALT elevations and/or anti-HBc and HCV + R individuals without these alterations (Mann-Whitney U test, significance defined as p
