Lymphocyte immunophenotypes among anti-HTLV-I/II-positive blood donors and recipients. The Transfusion Safety Study Group.

The Transfusion Safety Study retrospectively screened a repository of serum specimens collected in late 1984-early 1985 to identify blood donors with antibody to human T-cell lymphotropic virus (HTLV) at that time. They and their recipients have been traced for additional HTLV studies. Immunophenotypic analyses of peripheral blood lymphocytes from nine anti-HTLV-positive recipients, assumed to be infected during or since late 1984, showed no significant changes from healthy controls. Evaluation of the immunophenotypes of the 48 donors, however, showed significant elevations in the absolute counts of the T-cell (CD2) and natural killer (CD56) populations, the T helper/inducer and suppressor/inducer subsets (CD4+ CD29+ and CD4+ CD45RA+), and changes in T-cell activation markers. Long-term but not recent HTLV infection appears to alter the T-cell immunophenotypic pattern. Both infection with HTLV and human immunodeficiency virus type 1 are associated with a decreased CD2+ CD26+ count.

Risk of human immunodeficiency virus type 1 infection among sexual and nonsexual household contacts of persons with congenital clotting disorders.

The status of human immunodeficiency virus type 1 (HIV-1) infection at the time of transmission to sexual contacts remains poorly defined. Transmission to nonsexual household contacts has appeared to be rare. A total of 505 sexual and nonsexual contacts of HIV-1-infected hemophiliacs in 349 households was observed. At entry, 10% of 201 sexual partners were anti-HIV-1-positive. Follow-up of 151 uninfected partners during a total of 351 person-years of observation showed no sero-conversions, although there were 13 pregnancies during that period. Eighty-seven percent of the seronegative respondents to a detailed questionnaire reported unprotected sexual contact at least occasionally. Among 304 other household members, including 108 parents who helped administer clotting factor concentrates to their children, none was seropositive at entry. Follow-up of 263 showed no seroconversions during a total of 605 person-years of observation. Thus, anti-HIV-1-positive hemophiliacs transmitted to their partners earlier in their course but were not found to do so when prospectively observed. No relationship to level of viremia as indicated by CD4 count, HIV-1 p24 antigenemia, or acquired immunodeficiency syndrome was found. Anti-HIV-1-positive hemophiliacs had not transmitted to their nonsexual household contacts before study entry and did not do so subsequently, indicating that the risk from even close nonsexual contact is extremely low.

Variability in serial CD4 counts and relation to progression of HIV-I infection to AIDS in haemophilic patients. Transfusion Safety Study Group.

OBJECTIVE--To examine the CD4 count and its near term changes relative to progression to AIDS within 30 months and to subsequent CD4 counts. DESIGN--Longitudinal clinical and laboratory study. SETTING--Haemophilia treatment centres in six large American cities. PATIENTS--555 people with congenital clotting disorders who were infected with HIV, initially without AIDS, and seen at follow up for 6-30 months in 1986-9. MAIN OUTCOME MEASURES--Absolute CD4 counts and incidence of AIDS. RESULTS--Outset CD4 count and age were independently related to progression to AIDS (p less than 0.0001 and p less than 0.005 respectively). Patients with CD4 counts of 0.30-0.49 x 10(9) cells/l had an age adjusted risk of AIDS within 30 months of only 9% that of patients with counts less than 0.20 x 10(9)/l. Children under 10 years old had only 16% of the CD4 adjusted risk of AIDS of people aged greater than or equal to 45 years. Analysis of 149 patients' CD4 counts at the beginning and end of two successive six month intervals showed an average decrease of 11% in each six months regardless of the outset count (greater than or equal to 0.20 x 10(9)/l). For individual patients the decrease in the second six month period was unaffected by the decrease in the first six month period. CONCLUSIONS--Antiviral treatment of asymptomatic people, particularly children, with CD4 counts greater than or equal to 0.3 x 10(9)/l is questionable if predicted on near term progression to AIDS. Because of individual CD4 count variability and the low rate of progression to AIDS near term declines in individual CD4 counts are a poor index for identifying people who will rapidly progress to AIDS.

Abnormal B-cell function in hemophiliacs treated with cryoprecipitate and factor VIII and IX concentrates.

B-Cell function was evaluated in a group of 43 patients with factor VIII or factor IX deficiency. Thirty had been treated primarily with cryoprecipitate and 13 with concentrates of factor VIII or IX. Serum immunoglobulin G levels were found to be diffusely elevated; however, the absolute number of mature B cells in peripheral blood was normal. B-Cell function as measured by testing mitogen-induced proliferation and and immunoglobulin secretion by plaque-forming cells (PFC) in vitro was reduced. Coculture experiments suggested that these abnormal B-cell responses might be secondary to increased suppressor T-cell activity, which was found more frequently in patients seropositive for antibody to lymphadenopathy-associated virus. Both seronegative and seropositive patients had reduced responses in the proliferative and PFC assays, but the lowest PFC responses occurred in the seropositive group.

Plasma viremia in human immunodeficiency virus infection.

To determine which markers of human immunodeficiency virus type 1 (HIV) replication correlate most closely with progressive disease, we compared the following: (1) the frequency of isolation of HIV from peripheral-blood mononuclear cells (PBMC), (2) the frequency of isolation of the virus from cell-free plasma (plasma viremia), (3) the presence and titer of p24 antigen in plasma, and (4) the presence and titer of antibody to p24 antigen. We studied 213 persons who were positive for HIV antibody and 71 who were negative. HIV was isolated from PBMC from 207 of the 213 antibody-positive patients (97 percent), regardless of the clinical stage of the infection. Plasma viremia, in contrast, was correlated with the clinical stage of the infection. It was detected in 11 of 48 patients (23 percent) with asymptomatic infection, 32 of 71 (45 percent) in Class IVa of the Centers for Disease Control (those with AIDS-related complex), and 75 of 92 (82 percent) in Class IVc (those with AIDS) (P less than 0.01). Plasma HIV titers ranged from 10(0) to 10(4.3) and rose from a mean of 10(1.4) in asymptomatic patients to 10(2.5) in those with AIDS (P less than 0.02). Only 45 percent of patients with plasma viremia had HIV p24 antigen in either serum or plasma, and no correlation was found between the amount of p24 antigen in plasma and the plasma HIV titers. Follow-up tests indicated that plasma viremia was associated with a more marked decline in the CD4-lymphocyte cell count and the development of symptomatic disease (P = 0.034). We conclude that plasma viremia is a more sensitive virologic marker of the clinical stage of HIV infection and viral replication than the presence of p24 antigen or antibody in plasma. Not only whole blood but cell-free plasma from HIV-infected patients should be considered potentially infectious.

Treatment type and amount influenced human immunodeficiency virus seroprevalence of patients with congenital bleeding disorders.

Two hundred and eighty-two patients with congenital bleeding disorders received blood component replacement therapy between January 1979 and April 1985, were followed-up by the Puget Sound Blood Center's Hemophilia Care Program, and were tested for antibody to human immunodeficiency virus (HIV). Serologic results were obtained at least 1 year after the last exposure to volunteer donor products that were prepared before donor HIV screening or after the last exposure to concentrates produced before the manufacturer's use of treatment methods for inactivation of HIV. In all, 106 patients were anti-HIV positive. The risk of HIV infection was greater in patients with more severe bleeding tendencies, greater exposure to components, and exposure to lyophilized concentrates from large pools of donors. Of 100 patients with hemophilia A who only received cryoprecipitate from volunteer donors from Washington State (during the 6.3-year period), 14% had become anti-HIV positive. Of 27 patients receiving mostly cryoprecipitate but also being exposed to a single lot of concentrate during the same period, 13 (48%) were positive. Of 49 patients treated predominantly or solely with factor VIII concentrates during this period, 43 (88%) were anti-HIV positive. Of 29 patients with von Willebrand disease, four were anti-HIV positive, including 2 of 26 receiving only cryoprecipitate and two of three who had received a single dose of factor VIII concentrate. Of 19 patients who were treated solely with volunteer donor plasma, all remained anti-HIV negative. Of 47 patients exposed to factor IX concentrate, 28 (60%) were positive. Data relevant to the risk of HIV transmission subsequent to screening of the volunteer donor population were also obtained. Treatment records of 55 hemophilia A patients who have remained anti-HIV negative through at least June 1990 showed exposure to 71,173 screened donors from May 1985 through December 1989, and all 55 patients have remained anti-HIV negative.

Immunologic status of hemophilia patients treated with cryoprecipitate or lyophilized concentrate.

We evaluated 37 patients with moderate or severe hemophilia A and six patients with severe factor IX deficiency for clinical or laboratory evidence of immune abnormalities. Patients were assigned to one of four groups according to the type of clotting factor replacement. Twenty patients had received only cryoprecipitate during the two years preceding the evaluation (group I); 11 additional patients were treated predominantly with cryoprecipitate but had also received up to nine bottles of factor VIII concentrate (group II); six patients received factor VIII concentrate (group III); six patients received factor IX concentrate (group IV). There was no clinical or laboratory evidence of immunodeficiency among the 43 patients. The mean absolute number of Th cells was normal in all patient groups, but the mean absolute number of Ts cells was increased compared with controls, both in patients treated with cryoprecipitate and in patients treated with factor VIII or factor IX concentrate. There was no correlation between the Th/Ts ratio and patient age, alanine aminotransferase level, hepatitis serology, in vitro lymphocyte function, or amount of clotting factor administered. Our observations demonstrate that the volunteer or commercial origin of clotting factor replacement cannot fully explain the alterations in lymphocyte subset distribution previously described in patients with hemophilia A.

Effect of age on human immunodeficiency virus type 1-induced changes in lymphocyte populations among persons with congenital clotting disorders. Transfusion Safety Study Group.

Children other than neonates infected with human immunodeficiency virus type 1 (HIV-1) have low rates of progression to acquired immunodeficiency syndrome (AIDS). Through 1989, 5.3% of 95 infected hemophiliacs aged 5 to 13 years developed AIDS, compared with 20.3% of 364 aged greater than or equal to 25 years. We asked whether the HIV-1 impact on peripheral blood mononuclear cell subpopulations differed with age using pairwise comparisons of uninfected and infected male children and adult hemophiliacs. Infected children had lesser reductions of total lymphocytes than adults, but proportionately lower numbers of CD2+, CD4+, CD2+CD26+, and CD4+CD29+ counts. CD4+CD45RA+ cell counts were greater than twofold higher in uninfected and infected children than adults; with infection, the CD4+CD45RA+/CD4+ proportion increased by 1.4-fold in adults, but was unchanged in children. Infected adults had highly significantly increased total CD8+ counts; both age groups had elevated CD8+HLA-DR+ counts. Infected children had significantly higher total B-cell counts than infected adults, with a disproportionately lower number of resting B cells (CD20+CD21+). During 2 years of follow-up, infected children and adults had lymphocyte changes in the same directions and these were proportionately equal. The lower rate of HIV-1 progression in children may be partly associated with differences in lymphocyte populations compared with adults; functional properties of immune cells may be equally or more important.

Lymphadenopathy-associated virus antibodies and T cells in hemophiliacs treated with cryoprecipitate or concentrate.

Evidence for exposure to lymphadenopathy-associated virus (LAV) was investigated in 48 patients with hemophilia, 15 of whom had been treated exclusively with single-donor cryoprecipitate. The prevalence of antibodies to LAV in all patients was 53% in 1983 and 63% in 1984, while in patients treated only with cryoprecipitate, the prevalence was 31% in 1983 and 40% in 1984. Patients treated with any concentrate had a seroprevalence of 65% in 1983 and 77% in 1984. Seropositive patients were more likely to have a significant reduction in the ratio of helper to suppressor T cells, absolute numbers of helper T cells, and T cell function in vitro. Seven of 18 patients who were seronegative in 1983 had seroconverted by 1984. The relative risk of seroconversion for patients using any concentrate since 1981 compared with those using cryoprecipitate only was 3.9 (P = .04). Nevertheless, the rate of conversion in the latter group was 18% per year.

Rapid freezing of whole blood or buffy coat samples for polymerase chain reaction and cell culture analysis: application to detection of human immunodeficiency virus in blood donor and recipient repositories. The Transfusion Safety Study Group.

Storage of lymphocytes for later use in prospective epidemiologic studies of blood donors and transfusion recipients has been limited by the cost of separating peripheral blood mononuclear cells (PBMCs). When the Transfusion Safety Study began in 1985, it was decided to establish a cell repository of cryopreserved buffy coat (BC) samples, and thus far over 20,000 samples have been accumulated from enrolled subjects. To determine if these specimens could be used for polymerase chain reaction, a simple thawing and pelleting technique for recovering hemoglobin-free total white cells (WBCs) was developed. To validate the technique, parallel analysis was conducted of BCs, whole blood (WB), and PBMC samples from human immunodeficiency virus type 1 (HIV-1)-seropositive subjects. Immediate postthaw cell courts of 29 frozen-thawed (F-T) WB and BC samples averaged 90 percent of the prefreeze (input) values. Representative WBC populations were obtained by immediate pelleting. Amplification of HIV-1 gag sequences from F-T BCs and F-T WB was 94 and 75 percent, respectively, which is as sensitive as that obtained with freshly separated PBMC lysates. Quantitative HIV-1 proviral load analysis by serial dilution of 23 F-T BCs and 8 WB lysates showed results comparable to those obtained with lysates of fresh PBMCs. Values for WBC differential and immunophenotyping could be applied to express viral load relative to total WBCs, PBMCs, or CD4+ cells. These results establish the basis for simplified virologic analysis of cryopreserved BC or WB specimens.

Transmission of human immunodeficiency virus type 1 by dry-heated clotting factor concentrates. Transfusion Safety Study Group.

The Transfusion Safety Study monitored susceptible persons for human immunodeficiency virus type 1 (HIV-1) infections transmitted by plasma products and blood components. Through December, 1988, 6 subjects without antibody to HIV-1 (anti-HIV-1) became seropositive after receiving dry-heated factor VIII concentrate. The preparations implicated in 3 cases were derived entirely from anti-HIV-1-screened donors. In all instances, HIV-1 infection could be explained by concentrates heated at 60 degrees C for 24-30 h. Limiting consideration to concentrates and components administered after study entry showed that 4 of the seroconversions occurred among 122 subjects given 10 million units of factor VIII concentrates. No seroconversions occurred among 84 subjects given 5 million units of factor IX concentrates, or 83 who received components from over 26,000 unpaid donations. Serologic surveillance of anti-HIV-1-negative subjects provides important information, and should be routine in the management of persons receiving clotting factor concentrates.

Leukocyte immunophenotyping by flow cytometry in a multisite study: standardization, quality control, and normal values in the Transfusion Safety Study. The Transfusion Safety Study Group.

The Transfusion Safety Study (TSS) is a multicenter, cooperative investigation of factors that may determine the occurrence and modify the expression of transfusion-transmitted infections. A flow cytometry laboratory was established in each of the six participating centers in order to avoid alterations in cell phenotypes which may be caused by shipping delays, temperature changes, and handling. As a consequence, in order to assure compatibility of results, stringent standardization, quality control, and proficiency testing procedures were developed. This paper documents (i) the effect of time from phlebotomy to specimen staining and then to analysis for the antibodies used in the study; (ii) the effects of variations in light scatter cursor location for certain antibodies; (iii) a quality control program and data management and analysis system, each specifically designed for the study; and (iv) presents extensive data on age- and sex-related reference (normal) ranges for the several individual and paired monoclonal antibodies used in the study. Problems encountered, including obtaining reliable absolute lymphocyte counts, interference by nucleated erythrocytes, and sources of variability in results, are discussed. This study is meant to serve as a reference for future TSS publications.

Effect on lymphocyte subsets of clotting factor therapy in human immunodeficiency virus-1-negative congenital clotting disorders. The Transfusion Safety Study Group.

Patients with hemophilia A without human immunodeficiency virus type 1 (HIV-1) infection have lower CD4+ counts and CD4+/CD8+ ratios than controls. This is usually interpreted as a therapy-induced immunodeficiency. Our data re-examine the effect of therapy on peripheral blood mononuclear cell immunophenotypic subpopulations in all congenital clotting disorders. Since late 1985 we have prospectively observed HIV-1 uninfected persons with all types and severity of disorder. Controls were household members without clotting disorders or HIV-1 infection. Analyses of immunophenotype and treatment included a longitudinal random effects model. Compared with controls, age-adjusted CD4+ counts were significantly lower in treated patients (P < .0001) and in patients with all types of clotting disorders who were seldom or never treated (P = .0005). Significantly lower values among both treated and untreated clotting disorder subjects (P < .05) were likewise found for total lymphocytes, several other T-cell subsets, and the CD4+/CD8+ ratio. For most indexes, including the CD4+ count and CD4+/CD8+ ratio, the type of clotting deficiency was not a significant variable. Comparing persons who had no or minimal therapy with those having the most showed increases in CD8+ (P = .0017) and CD20+ CD21- counts (P = .0255), and a lower CD20+ CD21+/CD20+ ratio (P = .0106) in the latter. Controls and persons with clotting disorders differ in CD4+ count. Among those with clotting factor disorders, there is no difference attributable to type of clotting disorder or factor therapy. Large amounts of treatment increased CD8+ and CD20+ CD21- counts, but were not associated with a change in CD4+ count.

Effect of low- and intermediate-purity clotting factor therapy on progression of human immunodeficiency virus infection in congenital clotting disorders. Transfusion Safety Study Group.

Low- and intermediate-purity clotting-factor therapies are believed to accelerate human immunodeficiency virus (HIV) progression in hemophiliacs through adverse immune effects of the other plasma proteins in the preparations. To investigate this postulate, we evaluated data from six clinical centers that observed persons with congenital factor deficiencies at 6-month intervals. The present analysis is based on HIV-infected subjects who received intermediate purity factor VIII or factor IX concentrates, or cryoprecipitate. For long-term outcome, we classified 374 subjects by the type and amount of treatment during our first year of observation, and determined the subsequent rate of progression to a CD4 count less than 200 cells/microL. A second analysis of this group used a repeated-measures, random-effect model that allowed for individual differences in CD4 decline. Finally, we compared short-term rates of change in CD4 count in each treatment interval of 525 subjects with the type and amount of factor therapy received in the same interval. There was no overall or dose-related deleterious effect of any form of treatment on CD4 trend. The CD4 decrease was less when cryoprecipitate was administered alone or combined with concentrate, but not significantly so. Our results counter the assertion that low- and intermediate-purity products accelerate the rate of CD4 decrease in HIV-1-infected hemophiliacs.

Screening of blood donors for idiopathic CD4+ T-lymphocytopenia.

BACKGROUND: The recent recognition of idiopathic CD4+ T-lymphocytopenia (ICL) had led to concern that an unknown immunodeficiency virus may be transmissible by transfusion. STUDY DESIGN AND METHODS: To evaluate the prevalence and significance of low CD4+ values among blood donors, CD4+ data on 2030 blood donors who were negative for antibody to human immunodeficiency virus type 1 (HIV-1) were compiled. Those with CD4+ values below ICL cutoffs (< 300 CD4+ T cells/microL, or < 20% CD4+ T cells) were recalled for follow-up investigations. Serial CD4+ data on 55 homosexual men who seroconverted during prospective follow-up and data on 139 anti-HIV-1-positive blood donors initially evaluated in 1986 were reviewed as well. RESULTS: Five seronegative donors (0.25%) had absolute CD4+ counts < 300 cells per microL and/or < 20 percent. On follow-up, all five donors had immunologic findings within normal ranges, lacked HIV risk factors, and tested negative for HIV types 1 and 2 and human T-lymphotropic virus type I and II infections by antibody and polymerase chain reaction assays. Four of five donors reported transient illness shortly after their low CD4+ count donations. The median interval from HIV-1 seroconversion to an initial CD4+ value below ICL CD4+ cutoffs was 63 months for infected homosexual men. Of 139 HIV-1-infected blood donors studied 1 to 2 years after seropositive donations, 34 (24%) had CD4+ counts < 300 cells per microL and/or < 20 percent. CONCLUSION: Low CD4+ counts are rare among anti-HIV-1-negative volunteer blood donors and are generally associated with transient illnesses. If any unknown virus progresses similarly to HIV-1, CD4+ count donor screening would be a poor surrogate for its detection.

Randomized study of didanosine monotherapy and combination therapy with zidovudine in hemophilic and nonhemophilic subjects with asymptomatic human immunodeficiency virus-1 infection. AIDS Clinical Trial Groups.

To evaluate the safety and efficacy of didanosine (ddl) monotherapy and three different combinations of zidovudine (ZDV) and ddl in asymptomatic human immunodeficiency virus-1 (HIV-1) infection, we conducted an open-label, phase I/II study in 126 asymptomatic HIV-1-infected hemophilic and nonhemophilic subjects with a CD4 count of 200 to 500/mm3 stratified for prior zidovudine treatment and baseline CD4 count. Study arms included arm A, low-dose combination (ZDV 150 mg and ddl 134 mg, daily); arm B, moderate-dose combination (ZDV 300 mg and ddI 334 mg, daily); arm C, high-dose combination (ZDV 600 mg and ddl 500 mg, daily), and arm D, ddl monotherapy (ddl 500 mg, daily). Earlier, more frequent hepatotoxicity was experienced by hemophilic subjects (P = .008), but there were no differences in toxicity between treatment arms (P = .51), nor were there any differences in the rate of development of clinical endpoints by treatment (P = .41). Smaller median CD4 increases occurred over the first 12 weeks for arms A and D, 44/mm3 and 42/mm3, than arms B and C, 105/mm3 and 114/mm3, respectively, (P = .015). Hemophilia status (P = .0004) and prior ZDV experience (P = .044) independently predicted weaker CD4 responses during the first 12 weeks of treatment. Using a regression model and adjusting for hemophilia status, prior ZDV treatment, and baseline CD4, there was a significant reduction in quantitative viral load from baseline by week 12 for all treatment arms combined (P = .0001), with significantly lower median percent reduction for arm A (56.3%) than arms B, C, and D (94.6%, 98.5%, and 91.9%, respectively, P = .015). Although greater hepatoxicity and weaker CD4 responses occur in hemophilic subjects, didanosine monotherapy and combination therapy with zidovudine are safe and effective in asymptomatic HIV-1-infected patients.