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BACKGROUND: Persons with hemophilia (PWH) are at risk for chronic hemophilic arthropathy (HA). Joint replacement surgery may be used to relieve intractable pain and/or restore joint function. OBJECTIVES: This multicenter, prospective, observational cohort study evaluated the rate of bleeding during the postoperative period after total hip (THA) or knee arthroplasty (TKA). PATIENTS/METHODS: We included PWH of any severity ≥18 years of age who were undergoing THA or TKA. Clinical decisions were made at the discretion of the treating physician according to local standards of care. Clinical data were prospectively recorded. Major bleeding was defined as bleeding in a critical site, bleeding that resulted in either a 2 g/dL or greater decrease in hemoglobin during any 24-hour period, or transfusion of two or more units of packed red blood cells. RESULTS: One hundred thirty-one procedures (98 TKA and 33 THA) were performed, 39 (29.8%) of which were complicated by major bleeding, including 46% of THA and 25% of TKA. The risk of major bleeding was increased in THA compared to TKA (OR 2.50, p=0.05), and by the presence of an inhibitor (OR 4.29, p=0.04), increased BMI (OR 4.49 and 6.09 for overweight and obese, respectively, compared to normal BMI, each p

Background The goal of gene therapy for patients with hemophilia A is to safely impart long-term stable factor VIII expression that predictably ameliorates bleeding with the use of the lowest possible vector dose. Methods In this phase 1-2 trial, we infused an investigational adeno-associated viral (AAV) vector (SPK-8011) for hepatocyte expression of factor VIII in 18 men with hemophilia A. Four dose cohorts were enrolled; the lowest-dose cohort received a dose of 5 × 1011 vector genomes (vg) per kilogram of body weight, and the highest-dose cohort received 2 × 1012 vg per kilogram. Some participants received glucocorticoids within 52 weeks after vector administration either to prevent or to treat a presumed AAV capsid immune response. Trial objectives included evaluation of the safety and preliminary efficacy of SPK-8011 and of the expression and durability of factor VIII. Results The median safety observation period was 36.6 months (range, 5.5 to 50.3). A total of 33 treatment-related adverse events occurred in 8 participants; 17 events were vector-related, including 1 serious adverse event, and 16 were glucocorticoid-related. Two participants lost all factor VIII expression because of an anti-AAV capsid cellular immune response that was not sensitive to immune suppression. In the remaining 16 participants, factor VIII expression was maintained; 12 of these participants were followed for more than 2 years, and a one-stage factor VIII assay showed no apparent decrease in factor VIII activity over time (mean [±SD] factor VIII activity, 12.9±6.9% of the normal value at 26 to 52 weeks when the participants were not receiving glucocorticoids vs. 12.0±7.1% of the normal value at >52 weeks after vector administration; 95% confidence interval [CI], -2.4 to 0.6 for the difference between matched pairs). The participants had a 91.5% reduction (95% CI, 88.8 to 94.1) in the annualized bleeding rate (median rate, 8.5 events per year [range, 0 to 43.0] before vector administration vs. 0.3 events per year [range, 0 to 6.5] after vector administration). Conclusions Sustained factor VIII expression in 16 of 18 participants who received SPK-8011 permitted discontinuation of prophylaxis and a reduction in bleeding episodes. No major safety concerns were reported. (Funded by Spark Therapeutics and the National Heart, Lung, and Blood Institute; ClinicalTrials.gov numbers, NCT03003533 and NCT03432520.).

Background Factor VIII activity is determined by directly measuring the aPTT of a patient plasma sample and determining the percent activity from a standard curve generated from plotting the measured clotting time (in seconds) on a semi-log scale vs a known percent activity of the standard at several specific dilution points. Factor VIII activity for the patient samples is then performed on dilutions of patient plasma mixed with equal amounts of plasma deficient in the factor to be measured, and the percent of factor in the patient plasma is calculated from the standard curve by plotting the observed clotting time for a specific dilution of the patient sample. To minimize the potential for under-reporting an activity level or missing the presence of an inhibitor (defined as an antibody directed against Factor VIII), a subjective assessment of parallelism of the patient curve to the standard curve is performed. In absence of an inhibitor the standard and patient curves are parallel to each other with the patient curve’s slope (Ps) similar to the standard curve’s slope (Cs). In patients with an inhibitor, the clotting time is prolonged. Furthermore, with each subsequent dilution, the amount of inhibitor is diluted out, leading to shorter clotting times for subsequent dilutions. In practice this leads to a less steep patient curve slope (Ps) compared to the standard curve slope (Cs) and thus nonparallel lines. Aim Parallelism determination is currently a subjective assessment that leads to increased error in reporting, potential missed evaluation for inhibitors and potential unnecessary testing for inhibitors. We developed an objective and automated tool to assess parallelism as an added screening tool for the presence of a Factor VIII inhibitor. Methods We performed Factor VIII assays (Low Factor VIII assay modification on STA Compact using low curve calibration at 1:6, 1:15 and 1:30 dilution (STA Deficient VIII, Immuno-Depleted Plasma for Factor VIII:C assay by STA. Package insert 26217-revised September 1994)) at appropriate dilutions on Factor VIII deficient hemophilia patients. We examined curves for parallelism by comparing the ratio of the slope of the curve generated from patient dilutions without detectable inhibitor (disease-free state) and the slope of the curve generated from patient dilutions with a known inhibitor (disease state) vs the slope of the standard curve. We confirmed presence of an inhibitor for each patient sample by Bethesda assay utilizing the Nijmegen modification. Results Using a bell curve generated from a parameter simulation of obtained Ps/Cs ratios from screening 21 samples with and without an inhibitor, we determined a ratio of Ps/Cs of 0.45 to be a cut-off that was 100% sensitive and 80% specific for detection of an inhibitor. To confirm the validity of this cut-off, we screened 48 de-identified samples with and without low FVIII inhibitor (Bethesda titers Conclusion We developed and validated a new screening tool for detecting the presence of an inhibitor to Factor VIII during routine FVIII assays. This method has the potential to screen for the detection of inhibitors to other specific clotting factors such as FIX, lupus anticoagulants and the presence of the newer oral anticoagulants which directly inhibit Factor Xa or thrombin. Disclosures No relevant conflicts of interest to declare.

Men with hemophilia were initially thought to be protected from cardiovascular disease (CVD), but it is now clear that atherothrombotic events occur. The primary objective of the CVD in Hemophilia study was to determine the prevalence of CVD and CVD risk factors in US older men with moderate and severe hemophilia and to compare findings with those reported in age-comparable men in the Atherosclerosis Risk in Communities (ARIC) cohort. We hypothesized if lower factor levels are protective from CVD, we would see a difference in CVD rates between more severely affected and unaffected men. Beginning in October 2012, 200 patients with moderate or severe hemophilia A or B (factor VIII or IX level ≤ 5%), aged 54 to 73 years, were enrolled at 19 US hemophilia treatment centers. Data were collected from patient interview and medical records. A fasting blood sample and electrocardiogram (ECG) were obtained and assayed and read centrally. CVD was defined as any angina, any myocardial infarction by ECG or physician diagnosis, any self-reported nonhemorrhagic stroke or transient ischemic attack verified by physicians, or any history of coronary bypass graft surgery or coronary artery angioplasty. CVD risk factors were common in the population. Compared with men of similar age in the ARIC cohort, patients with hemophilia had significantly less CVD (15% vs 25.8%; P < .001). However, on an individual patient level, CVD events occur and efforts to prevent cardiovascular events are warranted. Few men were receiving secondary prophylaxis with low-dose aspirin, despite published opinion that it can be used safely in this patient population.

The development of neutralizing antibodies, referred to as inhibitors, against factor VIII is a major complication associated with FVIII infusion therapy for the treatment of hemophilia A (HA). Previous studies have shown that a subset of HA patients and a low percentage of healthy individuals harbor non-neutralizing anti-FVIII antibodies that do not elicit the clinical manifestations associated with inhibitor development.To assess HA patients' anti-FVIII antibody profiles as potential predictors of clinical outcomes.A fluorescence immunoassay (FLI) was used to detect anti-FVIII antibodies in 491 samples from 371 HA patients.Assessments of antibody profiles showed that the presence of anti-FVIII IgG1 , IgG2 or IgG4 correlated qualitatively and quantitatively with the presence of an FVIII inhibitor as determined with the Nijmegen-Bethesda assay (NBA). Forty-eight patients with a negative inhibitor history contributed serial samples to the study, including seven patients who had negative NBA titers initially and later converted to being NBA-positive. The FLI detected anti-FVIII IgG1 in five of those seven patients prior to their conversion to NBA-positive. Five of 15 serial-sample patients who had a negative inhibitor history and had anti-FVIII IgG1 later developed an inhibitor, as compared with two of 33 patients with a negative inhibitor history without anti-FVIII IgG1 .These data provide a rationale for future studies designed both to monitor the dynamics of anti-FVIII antibody profiles in HA patients as a potential predictor of future inhibitor development and to assess the value of the anti-FVIII FLI as a supplement to traditional inhibitor testing.

Men and boys who present with bleeding associated with low factor VIII levels and normal von Willebrand studies are assumed to have hemophilia A until proven otherwise. However, routinely available coagulation assays cannot easily distinguish mild hemophilia A from the 2N variant of von Willebrand disease. We present a case that highlights the difficulties of recognizing this diagnosis, the role of genetic testing, and the identification of a 2N variant that has not been previously described.

We studied the course of chronic HCV infections in a cohort of 222 persons with hemophilia (PWH) and von Willebrand disease followed at our center since 1973. Twenty two (10%) developed end stage liver disease (ESLD). Forty years after HCV infection, cumulative incidence of ESLD was 12.3% and overall survival was 45.5%. Those who were infected with HCV only (n = 100) had a survival of 75.2%, while those infected with HIV (n = 122) had a survival of 24% (P 0.001). Survivals were significantly longer for those infected with HCV at younger age (15 years) compared to those infected over age 30 years (P = 0.014). Cause specific deaths for ESLD and bleeding were 8.8% and 8.3% respectively. For HIV negative subjects, the annual hazard of death from ESLD and bleeding was near zero for the first 10 years, and then rose slowly over the next 20 years to 0.4/100py for ESLD and 0.2/100py for bleeding. Sixty subjects completed 79 treatment episodes. Sustained viral response rates increased from 7/21 (33%) between 1990 and 2001, to 17/29 (58%) between 2002 and 2011, and to 27/29 (93%), since 2012 with the advent of the directly acting antiviral agents. These results confirm the very slow ESLD progression rate in HIV negative PWH. However, the risk of death from both ESLD and bleeding increases steadily with longer duration of HCV infection. More aggressive surveillance to detect those with early fibrosis is needed now that curative treatment is possible in95% of individuals. Am. J. Hematol. 91:E335-E340, 2016. © 2016 Wiley Periodicals, Inc.

SummaryPerioperative clotting factor replacement is administered to reverse the inherent haemostatic defect in persons with haemophilia (PWH), potentially increasing their risk for developing venous thromboembolism (VTE) postoperatively. It was our objective to determine the prevalence of VTE in PWH undergoing total hip or knee arthroplasty (THA, TKA). Patients with haemophilia A or B who underwent THA or TKA were enrolled in this prospective, multicentre observational cohort study. Lower extremity venous duplex ultrasound was performed prior to surgery and 4–6 weeks after surgery. Eleven centres enrolled 51 subjects, 46 of whom completed the study. Six subjects (13.0 %) were treated with bypass agents perioperatively; the remaining 40 subjects received factor VIII or IX replacement. Intermittent pneumatic compression devices were utilised postoperatively in 23 subjects (50 %), and four subjects (8.7 %) also received low-molecular-weight heparin prophylaxis. One subject (2.2 %) with moderate haemophilia A was diagnosed with symptomatic distal deep-vein thrombosis (DVT) on day 6 following TKA. One subject (2.2 %) with severe haemophilia A was diagnosed with pulmonary embolism on day 9 following bilateral TKA. No subjects had asymptomatic DVT. Eighteen subjects (39.1 %) had major bleeding, and three subjects (6.5 %) experienced minor bleeding. The observed prevalence of ultrasound-detectable, asymptomatic DVT in PWH following TKA or THA in this study was low, but the incidence of symptomatic VTE (4.3 %, 95 % CI, 0.5–14.8 %) appeared similar to the estimated incidence in the general population without thromboprophylaxis.

Hepatitis C virus (HCV) treatment response rates remain low in HCV/HIV-1-coinfected individuals compared with those with HCV alone. Persons with inherited coagulation disorders have high rates of HCV and HIV-1 infection, but HCV treatment trials in this patient population are scarce. We hypothesized that differences by infection status in HCV viral kinetics would be associated with differences in HCV quasispecies complexity over time and with treatment response disparities. Coinfected and monoinfected patients were enrolled in a treatment trial for pegylated-interferon alpha-2a (peg-IFN) + ribavirin. Patients were treated for 48 weeks and followed for an additional 24. Quantitative HCV RNA was tested at multiple times during and after treatment. Viral kinetic parameters associated with response were estimated with a mathematical model. Quasispecies emergence was determined via heteroduplex complexity assay. Twenty-two patients were HCV RNA-positive at baseline, with no significant demographic or virological differences by infection status. Five of eleven (45%) of monoinfected and 3 of 11 (27%) of coinfected patients achieved sustained viral response (SVR). Peg-IFN efficacy (epsilon) of 90% or greater was associated with probability of end-of-treatment response (ETR) (P = .001) and SVR (P = .06). Patients with SVR had lower baseline quasispecies complexity than those without SVR (P = .07). Those with epsilon of 90% or greater also had lower baseline complexity (P = .07). Coinfection status mediated changes in complexity over time (P = .04). In conclusion, low pretreatment quasispecies complexity may predict peg-IFN response; early peg-IFN response is critical for sustained HCV clearance and is altered in coinfection. Further studies are warranted.

Before the mid-1980s, haemophilia often was unknowingly treated with contaminated plasma products, resulting in high rates of human immunodeficiency virus (HIV-1), hepatitis C virus (HCV) and hepatitis B virus (HBV) infections. To estimate the impact of these infections, a new cohort was established. All HCV-seropositive patients, age 13-88 years, at 52 comprehensive haemophilia treatment centres were eligible. Cross-sectional data collected during April 2001 to January 2004 (median June 2002) were analysed. Plasma HIV-1 and HCV RNA were quantified by polymerase chain reaction. Highly active antiretroviral therapy (HAART) was defined as use of at least three recommended medications. Among 2069 participants, 620 (30%) had HIV-1. Of 1955 with known HBV status, 814 (42%) had resolved HBV and 90 (4.6%) were HBV carriers. Although 80% of the HIV-1-positive participants had > or = 200 CD4+ cells microL(-1), only 59% were on HAART. HIV-1 RNA was undetectable in 23% of those not taking antiretroviral medications. Most (72%) participants had received no anti-HCV therapy. HCV RNA was detected less frequently (59%) among participants treated with standard interferon plus ribavirin (P = 0.0001) and more frequently among HIV-1-positive than HIV-1-negative participants (85% vs. 70%, P < 0.0001). HIV-1-positive participants were more likely to have pancytopenia and subclinical hepatic abnormalities, as well as persistent jaundice, hepatomegaly, splenomegaly and ascites. HAART recipients did not differ from HIV-negative participants in the prevalence of ascites. The clinical abnormalities were more prevalent with older age but were not confounded by HBV status or self-reported alcohol consumption. Eleven participants presented with or previously had hepatocellular carcinoma or non-Hodgkin lymphoma. Although prospective analysis is needed, our data reveal the scale of hepatic and haematological disease that is likely to manifest in the adult haemophilic population during the coming years unless most of them are successfully treated for HIV-1, HCV or both.

In hepatitis C virus (HCV) infection, virus load and the risk for HCV-related end-stage liver disease (ESLD) are increased among persons with human immunodeficiency virus (HIV) coinfection. To clarify these relationships, 42 hemophilic patients who developed ESLD and random samples from 164 hemophilic patients with HCV infection alone and 146 with HCV-HIV coinfection were tested for HCV load and genotype. HCV genotype was unrelated to HIV and age. In contrast, HCV load was higher with older age (P(trend)=.0001) and with HIV coinfection (6.2 vs. 5.9 log(10) genome equivalents/mL, P=.0001). During 16 years of follow-up of dually infected patients, ESLD risk was unrelated to HCV load overall (P(trend)=.64) or separately to HCV genotype 1 and genotypes 2 or 3 (P(trend)> or =.70). Irrespective of virus load, incidence of ESLD was marginally increased 2-fold (95% confidence interval, 0.8-5.6) with HCV genotype 1. Understanding the discordance between HCV load and ESLD, despite HIV's link to each of these, may help clarify the pathogenesis of HCV-related disease.

We recently described 102 HIV-1 integrase sequences that were amplified from blood cells or plasma obtained up to 18 years ago from 5 hemophiliacs who later died of AIDS and 5 hemophiliacs subsequently classified as slow or nonprogressors ( J Acquir Immune Defic Syndr Hum Retrovirol 1998;19:99-110). Although the region of the HIV-1 genome that encodes integrase was highly conserved, none of the deduced protein sequences of the patient-derived enzymes matched that of the clade B consensus or standard laboratory integrases. To test the hypothesis that the activity of HIV-1 integrases prevalent within an infected person contributes to the rate of disease progression, we have now expressed and purified these proteins and compared them in various assays. Most of the 75 unique full-length integrase proteins from the 102 clones were enzymatically active. Comparison of proteins derived from samples obtained soon after infection showed that the specificity and extent of viral DNA processing and the amount of DNA joining (the two biologically relevant activities of integrase) did not differ between the two groups of patients. In addition, the relative usage of alternative nucleophiles for processing and the amount of nonspecific nicking catalyzed by the proteins were indistinguishable between the patient groups. Although the patient-derived enzymes often exhibited different patterns of target site preferences compared with the laboratory integrase, there was no correlation with clinical course. Thus, the activities of HIV-1 integrases prevalent within these infected individuals, at least as reflected by standard assays, did not influence or predict the rate of disease progression.

Inhibitors are a rare but serious complication of treatment of patients with haemophilia. Phase III clinical trials enrol too few patients to adequately assess new product inhibitor risk. This project explores the feasibility of using a public health surveillance system to conduct national surveillance for inhibitors. Staff at 17 U.S. haemophilia treatment centres (HTC) enrolled patients with haemophilia A and B into this prospective study. HTC staff provided detailed historic data on product use and inhibitors at baseline, and postenrolment patients provided monthly detailed infusion logs. A central laboratory performed inhibitor tests on blood specimens that were collected at baseline, annually, prior to any planned product switch or when clinically indicated. The central laboratory also performed genotyping of all enrolled patients. From January 2006 through June 2012, 1163 patients were enrolled and followed up for 3329 person-years. A total of 3048 inhibitor tests were performed and 23 new factor VIII inhibitors were identified, 61% of which were not clinically apparent. Infusion logs were submitted for 113,205 exposure days. Genotyping revealed 431 distinct mutations causing haemophilia, 151 of which had not previously been reported elsewhere in the world. This study provided critical information about the practical issues that must be addressed to successfully implement national inhibitor surveillance. Centralized testing with routine monitoring and confirmation of locally identified inhibitors will provide valid and representative data with which to evaluate inhibitor incidence and prevalence, monitor trends in occurrence rates and identify potential inhibitor outbreaks associated with products.

Objective: To identify correlates of spontaneous hepatitis C virus (HCV) clearance among people with human immunodeficiency virus (HIV) co-infection. Design: Baseline (2001–2004) analysis of a cohort study of people with hemophilia. Methods: Detailed questionnaire data were used to identify dates of primary HCV and HIV infections and to categorize sex; race; alcohol use; interferon treatment; hepatitis B virus (HBV) status; and HIV/AIDS history, treatment and current status. Spontaneous HCV clearance was defined as nondetection of HCV RNA by polymerase chain reaction assay in paired annual plasma, excluding those treated with interferon. Chi-squared, Fisher exact test, and logistic regression were used to identify correlates of clearance. Results: Among 478 HIV-infected participants, 61 (12.8%) had cleared HCV. Among the 31 participants with chronic HBV (as well as HIV), 16 (51.6%) had cleared HCV. With chronic HBV, HCV clearance was increased 11.2-fold (95% confidence interval, 5.1–24.8), after adjusting for sex, race, and hemophilia severity. Excluding the participants with chronic HBV, the prevalence of HCV clearance was 10.1%; and it was significantly reduced among males (9.7%, P = 0.05), blacks (1.6%, P = 0.01), and participants with severe hemophilia (8.2%, P = 0.02). HCV clearance was not associated with HIV RNA detection in plasma, CD4 cell count, anti-HIV therapy, AIDS history, ages at or years of HIV or HCV infection, or alcohol consumption. Conclusions: HCV clearance is unambiguously and markedly increased with chronic HBV infection among HIV co-infected people.

Patients with inherited bleeding disorders who received clotting factor concentrates before 1987 have high rates of hepatitis C virus (HCV) or HCV/HIV infection. We evaluated HCV quasispecies evolution in longitudinally collected specimens comparing those from patients with progression to end-stage liver disease (ESLD; cases) to those with compensated chronic hepatitis (controls). Plasma samples were obtained from the National Cancer Institute Multicenter Hemophilia Cohort Study. Controls were matched for age, sex, infection duration, and presence/absence of HIV. Samples from early infection were compared to those obtained after onset of ESLD in the cases. The first hypervariable (HVR1) and core proteincoding regions were amplified, subcloned, and sequenced. Complexity and diversity were determined. More than 700 sub-clones from 10 pairs of patients (8 with HIV) followed over approximately 9.3 years were evaluated. HVR1 complexity narrowed over time in the cases, whereas it increased in controls (P = .01). Similar trends were observed for diversity within HVR1 and the core region (P = .04). HCV-infected patients with inherited bleeding disorders undergo quasispecies evolution over time. Evolution patterns differ for progressors and nonprogressors. Further understanding of these mechanisms may help identify factors related to progression rate and treatment response.

Many persons with hemophilia were infected with hepatitis C and B viruses (HCV, HBV) and HIV, but the consequences of these transfusion-acquired infections are poorly defined. We estimated the risk of HCV-related end-stage liver disease (ESLD) and the associations of age, HBV, and HIV with that risk. All 1816 HCV-seropositive hemophilic patients at 16 centers were followed for up to 16 years. Of these, 624 were HIV(-) and 1192 were HIV-coinfected; 135 had persistent HBV surface antigenemia, 1374 had resolved HBV infection, and 287 were HBV-uninfected. ESLD was defined as bleeding esophageal varices, hepatic encephalopathy, persistent ascites, or death excluding nonhepatic causes of these conditions. Competing risk models were used to estimate the annual hazard rate and cumulative incidence of ESLD. Proportional hazards models were used to estimate relative hazards of ESLD with covariates. ESLD developed in 127 of the HCV/HIV-coinfected participants, with an estimated 16-year cumulative incidence of 14.0% (95% confidence interval [CI], 11.6%-16.4%). Without HIV, 10 HCV-infected participants developed ESLD, for a significantly lower cumulative incidence of 2.6% (95% CI, 1.0%-4.3%, P.0001). ESLD risk increased steeply with age in both groups. With HIV, ESLD risk was increased 8.1-fold (95% CI, 1.9-35.2) with HBV surface antigenemia, 2.1-fold (95% CI, 1.3-3.3) with fewer than 0.2 x 10(9)/L (200/microL) CD4(+) lymphocytes, and 1.04-fold (95% CI, 1.03-1.06) per year of age. Thus, HIV is associated with a markedly increased risk of HCV-related ESLD for persons with hemophilia, particularly with HBV infection, low CD4(+) lymphocytes, or older age.

Mechanisms that protect most high-risk HIV-1 seronegative (HRSN) persons are not well understood. Among hemophiliacs from the Multicenter Hemophilia Cohort Study who remained HIV-1 seronegative despite a high (94%) risk for acquisition of HIV-1 infection, only 7/43 were homozygous for the protective CCR5 Delta32 polymorphism. Among the remainder, neither CCR5 density nor beta-chemokine production, nor in vitro susceptibility to infection with the HIV-1 isolate JR-FL could distinguish HRSN hemophiliacs from healthy controls. When compared to lymphocytes of healthy controls not at risk for HIV-1 infection, diminished spontaneous lymphocyte proliferation was seen in lymphocytes of HRSN hemophiliacs as well as in lymphocytes of hemophiliacs not at risk for HIV-1 infection. Surprisingly sera/plasmas obtained from high-risk HIV-1 seropositve hemophiliacs prior to seroconversion more often contained alloreactive antibodies than date-matched sera/plasmas obtained from HRSN hemophiliacs. Thus alloreactivity may predispose to acquisition of HIV-1 infection after parenteral exposure.

Healthy male subjects received, 1 wk apart, single oral doses of epsilon-aminocaproic acid (EACA) 100 mg/kg alone, EACA with probenecid (0.5 gm), or EACA 2 hr after 2.0 gm probenecid. Probenecid (2.0 gm) reduced the 8-hr urinary clearance and recovery of EACA by 50% without affecting plasma kinetics. Recovery of EACA in urine rose to 78% of the dose 48 hr after EACA. Plasma clearance of EACA did not differ from control EACA urinary clearance when 0.5 gm probenecid was given with EACA. In both cases all the EACA dose was recovered in urine within 8 hr. Clinical Pharmacology and Therapeutics (1980) 28, 223–228; doi:10.1038/clpt.1980.154

The incidence of the acquired immune deficiency syndrome (AIDS) among persons infected with human T-lymphotropic virus type III (HTLV-III) was evaluated prospectively among 725 persons who were at high risk of AIDS and had enrolled before October 1982 in cohort studies of homosexual men, parenteral drug users, and hemophiliacs. A total of 276 (38.1 percent) of the subjects were either HTLV-III seropositive at enrollment or developed HTLV-III antibodies subsequently. AIDS had developed in 28 (10.1 percent) of the seropositive subjects before August 1985. By actuarial survival calculations, the 3-year incidence of AIDS among all HTLV-III seropositive subjects was 34.2 percent in the cohort of homosexual men in Manhattan, New York, and 14.9 percent (range 8.0 to 17.2 percent) in the four other cohorts. Out of 117 subjects followed for a mean of 31 months after documented seroconversion, five (all hemophiliacs) developed AIDS 28 to 62 months after the estimated date of seroconversion, supporting the hypothesis that there is a long latency between acquisition of viral infection and the development of clinical AIDS. This long latency could account for the significantly higher AIDS incidence in the New York cohort compared with other cohorts if the virus entered the New York homosexual population before it entered the populations from which the other cohorts were drawn. However, risk of AIDS development in different populations may also depend on the presence of as yet unidentified cofactors.

To evaluate the clinical impression that hemophiliacs express a high level of interest in physical activities associated with a significant risk of major bleeding, a self-administered questionnaire was given to the 17 hemophilic adolescents 11–19 years of age in a state hemophilia program, and to 17 age-matched non-hemophilic adolescents. Scores for risk-taking attitudes, anticipated risk-taking behavior and their apparent likelihood of carrying out the behaviors were calculated for 23 activities. Compared with controls, hemophiliacs neither express greater risk-taking attitudes, anticipate greater risk-taking behavior, nor appear more likely to carry out the behavior. Hemophiliacs 15–19 years of age appear more likely to carry out the stated risk-taking behaviors when compared with younger (11–14-year-old) hemophiliacs but not when compared with matched older non-hemophiliacs. Risk-taking activites selected by hemophiliacs tend to be adaptive for the limitations imposed by their illness. The conclusion that hemophiliacs appear more likely to engage in risk-taking behavior as they get older, although they express no greater overall interest in risk-taking than do non-hemophilic adolescents, needs to be confirmed by behavioral observations as home care programs improve the activity potential of young hemophiliacs.

Many homosexual men with the acquired immunodeficiency syndrome (AIDS) have an unusual acid-labile form of human leukocyte, or alpha, interferon in their serum. Male patients with classic hemophilia treated with lyophilized clotting-factor concentrates are also at high risk for the development of AIDS. To determine whether the level of alpha interferon may be a preclinical marker of early subclinical disease, we examined stored plasma and serum from three hemophilic patients with AIDS. Persistently elevated levels of the acid-labile form of alpha interferon were found in all three patients. In two patients the appearance of circulating alpha interferon preceded the onset of clinical disease by 3 to 10 months. In contrast, alpha-interferon levels were not elevated in 43 of 46 unselected patients with hemophilia; three of these patients had transient elevations. These results suggest that acid-labile alpha interferon may be a marker that can be used to identify affected asymptomatic members of high-risk groups before the onset of clinical disease.

Transaminase values [alanine amino transferase (ALT) and aspartate amino transferase (AST)] and markers for hepatitis B were serially determined in 558 hemophiliacs exposed to blood products. Hepatitis B surface antigen (HBsAg) persistent for over 12 months was present in 6% of the patients. Antibody to hepatitis B surface antigen (anti-HBs) was noted in 90% of the 259 patients treated with factor VIII or IX concentrates but in only 49% of the 43 patients treated with fresh frozen plasma (FFP) or cryoprecipitate. Persistently abnormal transaminase values were noted in 31% of the patients treated with commercial concentrates but in only one (2%) of the patients exposed to cryoprecipitate or FFP. This difference continued even when the two groups of patients were matched for the amount of blood products, up to 50, 000 units, which they had received in the study period. In the concentrate-treated patients, no correlation could be found between transaminase values and the number of units of factor VIII or IX they had received during the six years of the study (1973–1978).

The structural instability of red cells from hereditary spherocytic patients suggests that the primary molecular alteration in this disorder resides in the spectrin membrane skeleton, a macromolecular structure thought to control red cell shape. In this study, the protein composition of HS red cell ghosts, spectrin-depleted inverted vesicles, and spectrin heterodimers was quantitatively normal in 9 HS patients from 3 unrelated families. The binding of 32P-spectrin heterodimers to spectrin-depleted inverted vesicles (physiological ionic strength, pH 7.5, 4°C) indicated a KD of 18.6 ± 2.0 n m (mean ± s.e. ) and a maximal binding capacity of 98 ± 7 μg of spectrin bound/mg of inverted vesicle protein for 9 HS patients, and 18.3 ± 1.8 n m and 116 ± 8 μg spectrin/mg of inverted vesicle protein in 9 paired normal controls. Therefore the binding of spectrin to the syndeins (Bands 2.1 → 2.6) its high affinity membrane binding site, is unaltered in hereditary spherocytosis.

The fibrinolytic system was assessed in 28 hemophiliacs using the chromogenic substrate H-D-Val-Leu-Lys-pNA. To determine whether a state of hyperfibrinolysis might be associated with Factor VIII replacement therapy, 14 patients with severe disease who were intensively treated with Factor VIII concentrates were compared with 14 patients with mild disease who were receiving infrequent transfusions with cryoprecipitate or fresh frozen plasma. Seventeen normal males served as controls. With the exception of an elevated level of plasminogen activator and a decreased level of immediate antiplasmin in the mild group only, no evidence of enhanced fibrinolysis was found. Other components of the fibrinolytic system were either normal (plasmin) or increased (progressive antiplasmin containing both alpha 2PI and alpha 2M, and plasminogen). The elevated plasminogen levels were found only in the severe intensively transfused group. The elevated progressive antiplasmin levels were found in both groups of patients and did not appear to be related to transfusions. These findings do not support the concept of enhanced fibrinolysis associated with intensive Factor VIII replacement therapy.

In a closed internal medicine practice for homosexual men in Central Manhattan herpes zoster developed in 112 men between 1980 and mid-1986. In these patients the incidence of acquired immunodeficiency syndrome (AIDS) was high: Kaplan-Meier survival analysis indicated cumulative incidences of AIDS of 22.8% within 2 years after herpes zoster, 45.5% within 4 years, and an estimated 72.8% after 6 years. Severity of zoster (relative risk, RR = 4.6), degree of pain (RR = 3.4), and zoster of the cranial or cervical dermatomes (RR = 2.2) were all associated with a poor outcome. Oral thrush, oral hairy leucoplakia, amoebiasis, and superficial (tinea) fungal infections also indicated an increased risk of AIDS among zoster patients. Oral thrush and oral hairy leucoplakia manifestations were diagnosed an average of 1.2 and 1.1 years, respectively, after the diagnosis of herpes zoster; thus zoster is an early indicator of an impaired immunity. Herpes zoster can be used as a predictor of AIDS and in AIDS risk groups should be regarded as a poor prognostic sign.

Summary. We recently observed an increase in factor-VIII clot promoting activity as measured by a one-stage assay (VIII AHF1) in a haemophiliac with hepatitis. However, VIII AHF as measured by a two-stage assay (VIII AHF2) was 0.013 u/ml at a time when VIII AHF1 measured 0.38 u/ml. We then studied seven non-haemophiliacs with liver disease, and attempted to correlate the levels of VIII AHF1 and VIII AHF2 with factor VIII-like antigen (VIII AGN) as measured by quantitative immunoelectrophoresis. In four of the seven patients, disproportionate elevations of VIII AHF2 compared to VIII AHF1 were found. Furthermore, VIII AHF2 values correlated well with VIII AGN values. No such discrepancy was apparent in four normal control subjects. These findings emphasize the necessity for performing two-stage assays in haemophiliacs as well as non-haemophiliacs with liver disease to assess factor-VIII levels. In addition, they suggest that confirmation of the diagnosis of haemophilia may not be possible in the haemophiliac with hepatitis unless VIII AHF2 determinations are performed. The reason for the disparity between VIII AHF1 and VIII AHF2 levels is not apparent. However, the correlation of VIII AGN and VIII AHF2 levels in the non-haemophiliacs with liver disease provides further support for the concept that VIII AGN and VIII AHF are closely related or identical molecular entities.

The acquired immunodeficiency syndrome (AIDS) was first recognized among hemophiliacs in 1982. The authors have conducted investigations to determine the onset and incidence of AIDS among hemophiliacs and to determine trends in hemophilia mortality since the introduction of clotting-factor concentrates in the late 1960s. A survey of United States hemophilia treatment centers, supported by the Centers for Disease Control and the National Hemophilia Foundation, defined a population of hemophiliacs which was monitored for AIDS cases through June 1984. Death reports from the United States Vital Statistics System and from the hemophilia treatment center survey provided mortality trends for 1968-1979 and for 1978-1982, respectively. The results of these investigations demonstrate the following points. 1) The AIDS epidemic is a new and important cause of illness and mortality among hemophiliacs, although a very low incidence of AIDS among hemophiliacs prior to 1982 cannot be ruled out. 2) The AIDS cases who attended the surveyed hemophilia treatment centers were distributed throughout the United States and were older than hemophilia treatment center patients without AIDS. AIDS cases also used more lyophilized clotting-factor concentrate, but only a small number of cases were reported with this information. 3) Improved care for hemophilia, including the use of clotting-factor concentrates, dramatically reduced hemophilia mortality rates during the 1970s. 4) In 1982, hemorrhage was the major cause of death among hemophiliacs. Deaths from non-alcoholic liver disease were also increased. AIDS incidence among hemophilia treatment center attendees was stable at 0.6 cases per 1,000 hemophilia treatment center attendees per year during 1982 and 1983 but increased sharply to 5.4 cases per 1,000 during the first quarter of 1984.

Antihemophilic factor (AHF) concentrates contain debris which passes freely through the 170 μ filter routinely used for patient infusion. We have previously shown that this material is composed in part of IgG, fibrinoprotein and cold-insoluble globulin and that it is retained by a screen filter of 40 μ pore size. The purpose of this study was to investigate pulmonary effects of this particulate material in hemophiliac patients receiving frequent AHF infusions. Pulmonary function studies were performed before and after the infusion of a standard dose of AHF concentrate calculated to raise the in vivo factor VIII level to 50 per cent of normal. Using a standard 170 μ filter, five of six patients showed a significant decrease (p L CO SB ) 30 minutes and 6 hours after the infusion, with values returning to preinfusion levels by 48 hours. Slow vital capacity, forced vital capacity, maximum voluntary ventilation and a helium dilution residual lung volume remained normal in all patients. Two patients who were restudied using a 40 μ filter showed no significant change in the D L CO SB after the infusion. We conclude that lodging of particulate material in the pulmonary capillary bed was most likely responsible for the reduced D l CO sb . Since the defect was prevented by microfiltration, the use of a finer mesh filter than is currently recommended may be desirable for hemophiliac patients receiving large amounts of AHF concentrate at frequent intervals.

We evaluated a multicenter cohort of 1219 subjects with hemophilia or related disorders prospectively, focusing on 319 subjects with documented dates of seroconversion to human immunodeficiency virus type 1 (HIV-1). The incidence rate of the acquired immunodeficiency syndrome (AIDS) after seroconversion was 2.67 per 100 person-years and was directly related to age (from 0.83 in persons 1 to 11 years old up to 5.66 in persons 35 to 70 years old; Ptrend = 0.00003). The annual incidence of AIDS ranged from zero during the first year after seroconversion to 7 percent during the eighth year, with eight-year cumulative rates (+/- SE) of 13.3 +/- 5.3 percent for ages 1 to 17, 26.8 +/- 6.4 percent for ages 18 to 34, and 43.7 +/- 16.4 percent for ages 35 to 70. Serial immunologic and virologic markers (total numbers of CD4 lymphocytes, presence of serum interferon or HIV-1 p24 antigen, and low or absent serum levels of anti-p24 or anti-gp120) predicted a high risk for the subsequent development of AIDS. Adults 35 to 70 years old had a higher incidence of low CD4 counts than younger subjects (P less than or equal to 0.005), whereas adolescents had a low rate of anti-p24 loss (P = 0.0007) and subjects 1 to 17 years old had a lower incidence of AIDS after loss of anti-p24 (P = 0.03). These findings not only demonstrate that the risk of AIDS is related directly to age but also suggest that older adults are disproportionately affected during the earlier phases of HIV disease, that adolescents may have a low replication rate of HIV, and that children and adolescents may tolerate severe immunodeficiency better because they have fewer other infections or because of some unmeasured, age-dependent cofactor or immune alteration in the later phase of HIV disease.

In Pennsylvania, the prevalence of hemophilia is one per 10,000 males. Factor VIII deficiency is five times more frequent than Factor IX deficiency, and 34% of the patients have no relatives affected with the disease. The mean age is 23 years old, and 50% of the patients are less than 20 years old. Approximately one-third of the patients with Factor VIII deficiency and one fourth of the patients with Factor IX deficiency have levels of less than 0.01 mu/ml. By clinical criteria, 55% of those with Factor VIII deficiency are severe compared to 45% of those with Factor IX deficiency. Factor VIII-deficient patients are treated an average of 18 times per year compared to ten times per year for patients with Factor IX deficiency. Hemarthroses account for 70% of the hemorrhages treated and for 40% of the concentrate usage. Home therapy patients use an average of 45,950 Factor VIII units per year at a cost of ø170 per patient and their use accounts for 60% of the total Factor VIII usage of 1.7 million units. Less than five days per patient per year are lost from school or work because of bleeding, and patients are hospitalized for bleeding an average of only two to three days per patient year. Adverse immediate reactions to therapy are infrequent. Five percent of patients have persistence of the hepatitis B virus, and 7.5% have inhibitors. The mortality rate is 0.04% per year, with half of the deaths being hemophilia-related.

We examined a family pedigree in which retinitis pigmentosa and hemophilia A were inherited in an X-linked manner. Six female carriers were identified by electroretinography. Results of ophthalmoscopic examination were normal in two women, while four displayed marked variability in phenotypic expression. Six of seven males identified with retinitis pigmentosa had hemophilia A. One asymptomatic boy had a markedly abnormal electroretinogram despite normal ophthalmoscopic examination. Pedigree analysis showed a high recombination rate, which would be expected as these two genes are known to be at opposite arms of the X chromosome.

Healthy hemophiliacs receiving antihemophilic (AHF) concentrates have decreased T-helper/suppressor (H/S) ratios, similar to the changes observed in healthy homosexuals considered at risk for the acquired immune deficiency syndrome (AIDS). We present a hypothesis which focuses on the role of natural killer (NK) cells and elevated serum levels of alpha interferon (IFN alpha) in hemophiliacs with AIDS. This hypothesis suggests that alloantigens in AHF concentrates provide an important inducing event which compromises the immune system of the hemophilia patient and enhances his susceptibility to infection by a transmissible agent. It provides an explanation for the predominance of NK cells with T-cell markers, impaired NK cell function, elevated serum IFN alpha levels, and the deficit in T-helper/inducer cells in hemophilic patients with AIDS. Furthermore, it could account for the T-cell subset imbalance in relation to total AHF units transfused, and to the development of an inverted H/S ratio which is a constant feature of AIDS.

Proportionate twofold or greater increases in both Factor VIII procoagulant activity (VIII AHF) and Factor VIII-like antigen (VIII AGN) have been observed to follow the administration of adrenalin in normal individuals. Levels of factor VIII procoagulant or clot-promoting activity (VIII AHF) and VIII AGN were measured immediately before and one hour after the subcutaneous injection of adrenalin (0.35 ml) in ten adults with hemophilia A documented by low VIII AHF levels (ranging from 0.01-0.25 units/ml, normal VIII AGN levels, and normal VIII VWF levels as measured by the washed-platelet ristocetin assay). Eighteen normal adults served as controls. Results were analyzed by the paired t test. It was shown that VIII AGN increased significantly, while VIII AHF did not change, in hemophiliacs after adrenalin stimulation, regardless of the base line level. This fixed rate of VIII AHF production, release, or activation may help to explain the constancy of VIII AHF levels in individual hemophiliacs and their affected family members.

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Excerpt To the editor: In a 15-month period during which time 4 700 000 units of factor VIII were used by 70 hemophiliacs known to us, fifteen adverse reactions were reported. Excluding surgical us...