Determination of Total Biotin by Liquid Chromatography Coupled with Immunoaffinity Column Cleanup Extraction: Multilaboratory Testing, Final Action 2016.02.

Single- and multilaboratory testing data have provided systematic scientific evidence that a simple, selective, accurate, and precise method can be used as a potential candidate reference method for dispute resolution in determining total biotin in all forms of infant, adult, and/or pediatric formula. Using LC coupled with immunoaffinity column cleanup extraction, the method fully meets the intended purpose and applicability statement in AOAC Standard Method Performance Requirement 2014.005. The method was applied to a cross-section of infant formula and adult nutritional matrixes, and acceptable precision and accuracy were established. The analytical platform is inexpensive, and the method can be used in almost any laboratory worldwide with basic facilities. The immunoaffinity column cleanup extraction is the key step to successful analysis.

[Iodine delivery rate of different concentrations of iodine-containing contrast agents with rapid injection]. / Jodeinbringungsgeschwindigkeit verschieden konzentrierter Röntgenkontrastmittel bei schneller intravenöser Injektion.

PURPOSE: To determine how the concentration of iodinated contrast media for computed tomography studies affects the iodine delivery rate at various conditions. MATERIALS AND METHODS: Three nonionic, iodinated contrast agents that are marketed for computed tomography applications were administered through 1.1 and 1.3 mm large peripheral vein catheters into a vein phantom with room temperature and with pre-heating of the agents at 37 degrees C using a power injector. Each injection applied 40 ml of contrast medium at a flow rate of four to eight ml/s. The iodine concentration of Iopromide, Iomeprol, and Iodixanol varied between 300 and 400 mg iodine per cc. The power injector used a pressure limit of 21 bar. For each experiment, the maximum iodine deliver rate was calculated from the highest possible flow rate recorded. RESULTS: Larger vein catheters and the higher contrast temperature yielded the highest flow rates. With the higher iodine concentrations, viscosity limited the injection speed of the achievable pressure limit. The highest iodine delivery rate was 2560 mg/s, using an agent with 320 mg/ml iodine content. With the more concentrated agent, a maximal iodine delivery rate of 2400 mg/s was achieved. CONCLUSION: Very high iodinated contrast agent concentrations do not increase the iodine delivery rate of rapid peripheral intravenous injections, since the high viscosity of such agents causes the injection pressure to increase more than the higher iodine concentration would compensate for. With lower injection velocities, the higher viscosity of highly concentrated contrast agents may remain without practical consequences.

HIV-induced IL-6/IL-10 dysregulation of CD4 cells is associated with defective B cell help and autoantibody formation against CD4 cells.

To analyse CD4 cell cytokine secretion and helper/suppressor function at a clonal level we established 446 CD4+ T cell clones (TCC) in four healthy controls, three HIV- haemophilia patients, four CDC II,III and four CDC IV patients. Spontaneous TCC secretion of Th1 cytokines (IL-2, interferon-gamma (IFN-gamma)) and Th2 cytokines (IL-4, IL-6, IL-10) was determined by ELISA. TCC helper and suppressor functions were tested in a pokeweed mitogen (PWM)-stimulated allogeneic co-culture system using a reverse haemolytic plaque assay for assessment of B cell responses. There was a significant association of TCC surface marker expression (Leu-8, CD45RA) with TCC IL-6 secretion in healthy controls (P < 0.01), HIV- patients (P < or = 0.001) and CDC II,III patients (P < or = 0.01) but not in CDC IV patients. Likewise, TCC expression of Leu-8 and CD45RA was significantly associated with TCC suppressor function in healthy controls (P < or = 0.0005) but not in HIV-infected patients. A reduced TCC helper frequency (< or = 10% of TCC) and an enhanced TCC suppressor frequency (> 80% of TCC) were detected only in those HIV-infected patients who showed an excessively increased TCC IL-6 secretion (> 70% of TCC) together with a significantly diminished TCC IL-10 secretion (< or = 10% of TCC). CD4 cell autoantibodies also were found only in patients with this type of cytokine dysregulation. These data indicate that CD4 cell surface markers lose their functional relevance in HIV-infected patients. HIV-induced IL-6/IL-10 dysregulation of CD4+ T cells, i.e. the up-regulation of spontaneous IL-6 and down-regulation of spontaneous IL-10 secretion, appears to be involved in inducing CD4 helper defects and may promote autoantibody formation against CD4 cells.

Low risk of viral infection after administration of vapor-heated factor VII concentrate or factor IX complex in first-time recipients of blood components. International Factor Safety Study Group.

BACKGROUND: Vapor-heated human factor VII concentrate and human factor IX complex are both obtained from prothrombin complex, undergo similar methods of manufacture, and are subjected to an identical two-step vapor-heating process for virus inactivation. STUDY DESIGN AND METHODS: Intermediate-purity vapor-heated human factor VII concentrate and vapor-heated human factor IX complex were monitored for safety with regard to viral infection in the context of an International Factor Safety Study, a prospective study that follows the revised recommendations from the International Congress of Thrombosis and Hemostasis (ICTH). Because the rarity of the respective hereditary deficiencies would have made separate analyses unrealizable, the results were combined for the final analysis. Entry required that patients have no history of transfusion with any blood derivative. After the first infusion of the study drug, patients were monitored for 6 months for the development of non-A, non-B hepatitis (NANBH) and infection with hepatitis B virus (HBV) and for 15 months for infection with hepatitis C virus (HCV) and human immunodeficiency virus (HIV). An event was defined as a positive result on any test for any infection. An alanine aminotransferase level more than 2.5 times the upper limit of normal on two consecutive occasions was defined as an event for NANBH. HBV infection was monitored with tests for three different HBV markers: the HBV surface antigen, antibody against the HBV surface antigen, and antibody against HBV core antigen. HCV and HIV infection were monitored with tests for HCV and HIV antibodies. RESULTS: The 25 patients who completed the study (1 has not completed the study and 1 dropped out) received a total of 434 infusions comprising 17 different production lots of the concentrates. Twenty patients were analyzable for NANBH and 25 for HCV and HIV infection. Since most patients had been given HBV vaccination, only 4 patients were analyzable for this end point. None of the patients showed evidence of having developed an event. These data satisfy ICTH criteria when the products are considered together, but vapor-heated factor VII concentrate does not qualify alone because there were only five patients in this group. CONCLUSION: Vapor-heated factor VII concentrate and vapor-heated factor IX complex are associated with a low risk of viral infection. Preliminary results are also presented, indicating that the concentrates are safe with regard to inhibitor development.

Should clinical freedom be constrained in the name of self-sufficiency?

Clinical freedom should enable a physician to decide in a free and unbiased manner which is the most appropriate therapy to use for a particular patient. In order to implement the four aims of the German Haemophilia Society an average of 4-4.5 units of Factor VIII per capita of the general population per year is needed. At present European countries do not produce this amount, but to reduce the consumption of F VIII in therapy lowers treatment levels. Until plasma collection services in Europe can be expanded it is necessary that the additional, imported, sources of plasma are available, otherwise clinical freedom will be curtailed.

Pharmacokinetics of two pasteurized factor VIII concentrates by different and multicenter assays of factor VIII activity.

We assessed the pharmacokinetic characteristics of a new high-purity pasteurized FVIII concentrate in comparison with an intermediate purity pasteurized concentrate, produced by the same manufacturer. The study was designed as a cross-over single-dose pharmacokinetic investigation in 8 non-bleeding patients with severe hemophilia A. All patients were given 25 IU/kg of each of the two concentrates, with an interval of at least one week between the two administrations. Decay curves were assessed by collecting 10 serial blood samples over 36 hours following the end of infusion. The concentration of Factor VIII in blood samples was determined in triplicate in three different laboratories using each of the following assay methods: a one-stage clotting assay, a two-stage clotting assay, and a two-stage chromogenic-peptide substrate assay. All pharmacokinetic parameters were calculated by model-independent methods. The two products were found to differ significantly both in the clearance, which was on average 13.8% lower for Haemate P, and in the in-vivo recovery, which was 11.7% lower for Factor VIII:C P on the average. In comparison with previous pharmacokinetic data obtained from other heated Factor VIII concentrates, the clearance of Haemate P was found to be significantly slower, while the half-life of both products was longer. No differences were observed in the Vd-area. These findings indicate that the purification procedures to which both products are subjected do not increase the in-vivo rate of plasma disappearance of Factor VIII.

Low risk of viral infection after administration of vapor-heated factor VIII concentrate. International Investigator Group.

A multicenter prospective study was carried out to evaluate whether a vapor-heated factor VIII concentrate transmitted blood-borne viral infections over a surveillance period of 15 months. Thirty-five patients with hemophilia and von Willebrand disease who had never received any blood components were treated. Twenty-eight were analyzed and found not to have non-A, non-B hepatitis. Sera from 20 of these 28 patients were also tested for the antibody to the hepatitis C virus. None had sero-converted during the follow-up period. None of the patients analyzed developed markers of the hepatitis B virus (n = 17) or the human immunodeficiency virus (n = 31). This vapor-heated factor VIII concentrate carries a low risk of transmitting hepatitis and human immunodeficiency virus infection.

Soluble IL-2 receptor and tumour necrosis factor-alpha in plasma of haemophilia patients infected with HIV.

We measured plasma concentrations of soluble receptors for IL-2 (sIL-2R) and tumour necrosis factor-alpha (TNF-alpha) in 149 haemophilia patients. Soluble IL-2R levels were elevated in 37% of 62 HIV-seronegative patients (mean 570 +/- 27 U/ml versus 361 +/- 17 U/ml in the control group, P less than 0.0001), in 78% of 68 HIV-seropositive patients (928 +/- 49 U/ml, P less than 0.0001), and in 95% of 19 AIDS/ARC patients (1578 +/- 199 U/ml, P less than 0.0001 compared with controls and with HIV-seronegative patients; P less than 0.005 compared with HIV-seropositive asymptomatic patients). A negative correlation was observed between sIL-2R, relative and absolute numbers of CD4+ cells (P less than 0.0001), and CD4/CD8 ratios (P less than 0.0001). There was also a negative correlation between sIL-2R in plasma and the cellular expression of IL-2R (P less than 0.001). We found a significant association of sIL-2R and plasma neopterin (P less than 0.0001). With progression of the disease from HIV-seronegative to seropositive without symptoms and to full manifestation of AIDS/ARC, sIL-2R plasma levels increased. The highest levels were found at the time of diagnosis of AIDS/ARC, but the levels decreased again during the following 18 months. Eight per cent of HIV-seronegative patients, 32% of HIV-seropositive patients, and 24% of patients with AIDS/ARC had increased plasma TNF-alpha. We conclude that sIL-2R and TNF-alpha plasma levels are elevated in HIV-infected haemophilia patients and that sIL-2R is a marker for disease progression from asymptomatic HIV-seropositive to AIDS/ARC.

Financial assistance for HIV-infected persons with haemophilia worldwide.

In many countries we could note the dramatic developments in regard to financial assistance of HIV-infected haemophiliacs and their families since we distributed the first overview on this to all World Federation of Hemophilia member countries. As a result of the untiring efforts of the National Haemophilia Societies, infected persons and their families receive essential assistance in 15 member countries. It is hoped that this development will sway more and more member countries to consider demands for financial support and provide them with the arguments to succeed.

Reproducibility of one-stage, two-stage and chromogenic assays of factor VIII activity: a multi-center study.

This study was aimed at assessing the reproducibility of Factor VIII assays between different laboratories using the same reagents. A total of 176 post-dose plasma samples were obtained from 8 Italian subjects with hemophilia-A treated with a single dose of Factor VIII concentrates. Three laboratories (in FRG, Italy, and Sweden) participated in the study. Frozen aliquots of each sample were dispatched to each of the laboratories, where the aliquots were assayed using the same one-stage, two-stage and chromogenic methods. The one-stage and the chromogenic methods were well reproducible between the three centers: pairwise correlation analyses yielded r-values ranging from 0.88 to 0.91 for the one-stage method and from 0.91 to 0.96 for the chromogenic method. The agreement between these two assays was less evident in samples with activity below 200 IU/L in which the one-stage gave, on average, higher Factor VIII concentrations than those provided by the chromogenic method. The two-stage method was not well reproducible, and the pairwise r-values ranged from 0.48 to 0.73. Our study emphasises the need to develop multi-center quality control programs to verify the reproducibility of Factor VIII assays.

Autoantibodies against CD4 cells are associated with CD4 helper defects in human immunodeficiency virus-infected patients.

To investigate whether autoantibodies against CD4-positive lymphocytes might induce helper dysfunction, autoantibody formation and T-cell function was examined simultaneously in 61 hemophilia patients. Twenty patients were human immunodeficiency virus (HIV)-negative, 26 HIV-positive stage CDC II or III, and 15 were HIV-positive stage CDC IV. T lymphocytes, CD4-positive, or CD8-positive T subsets were cocultured with B lymphocytes and pokeweed mitogen (PWM) for 6 days and Ig-secreting cells were assessed in a reverse hemolytic plaque assay. The presence of IgM, IgG, C3d, or gp120 on the surface of T cells or T subsets was analyzed by flow cytometry. Autoantibodies against CD4-positive T cells were not detected in controls or HIV-negative patients, but were common in HIV-positive patients (20 of 41 patients). In patients with autoantibodies we found an increased incidence of CD4 helper defects (P less than .0001 in CDC II or III patients; P less than .02 in CDC IV patients). 12 of 13 patients with IgM autoantibodies and 4 of 4 with IgG autoantibodies showed CD4 helper defects. Complement fixation had no relevance. Autoantibody formation against CD4 cells was not due to increased in vivo B-cell stimulation (spontaneous plaque formation: 611 +/- 204 PFC/10(6) B cells in autoantibody-negative patients v 650 +/- 202 PFC/10(6) B cells in autoantibody-positive patients; not significant). Thus, our results suggest that autoantibody formation is not caused by a general state of in vivo B-cell activation. Rather, the production of autoantibodies appears to coincide with defects in B-cell proliferation or differentiation, as shown by reduced mitogen-stimulated B-cell responses in CDC II and III patients (P less than .05). Autoantibodies against CD4 cells appear to be involved in the pathogenesis of CD4 helper defects of HIV-infected patients.

Low risk for hepatitis C in hemophiliacs given a high-purity, pasteurized factor VIII concentrate. International Study Group.

STUDY OBJECTIVE: To assess whether the hepatitis B virus (HBV), the hepatitis C virus (HCV), and the human immunodeficiency virus (HIV) are transmitted to hemophiliacs by a high-purity factor VIII concentrate in which the method of virus inactivation is pasteurization. DESIGN: Hepatitis B virus markers, the antibody to HCV (anti-HCV), the antibody to HIV (anti-HIV), and aminotransferases were measured on serum samples collected before the first concentrate infusion and at regular time intervals thereafter. SETTING: Seventeen hemophilia centers in Italy, the Federal Republic of Germany, Belgium, Austria, and the Democratic Republic of Germany. PATIENTS: Twenty-nine patients with hemophilia A who had not received a previous transfusion with blood products and who had normal alanine and aspartate aminotransferases (ALT and AST) were included in the final analysis. MEASUREMENTS AND MAIN RESULTS: No patient became positive for anti-HCV or anti-HIV or developed sustained increases in aminotransferase levels. Similarly, none of the 15 unvaccinated patients developed markers of HBV infection. CONCLUSION: This prospective study conducted in previously untransfused hemophiliacs highly susceptible to developing post-transfusion hepatitis shows that a large-pool clotting factor concentrate treated with pasteurization carries a low risk for transmitting HCV, the major causative agent of post-transfusion hepatitis.

Absence of anti-human immunodeficiency virus types 1 and 2 seroconversion after the treatment of hemophilia A or von Willebrand's disease with pasteurized factor VIII concentrate.

Patients with hemophilia A or von Willebrand's disease who are treated with concentrated preparations of human factor VIII made from unscreened pooled plasma are at substantial risk of contracting human immunodeficiency virus (HIV) infection. The purpose of this study was to investigate whether by treating such patients with a pasteurized factor VIII concentrate that had been heated in aqueous solution at 60 degrees C for 10 hours, HIV infection could be avoided. Eleven hemophilia centers in the Federal Republic of Germany and two in Austria identified 155 eligible patients who had been treated exclusively with pasteurized factor VIII concentrate and had not received any other blood products. Between February 1979 and December 1986 they received a total of 15,916,260 IU of pasteurized factor VIII. The United States was the source of 80 percent of the plasma from which the concentrate was made. By September 1988, these 155 patients had been screened for antibody to HIV type 1 (anti-HIV-1) with a total of 657 tests; all were negative. Sixty-seven patients were also tested once for antibody to HIV type 2 (anti-HIV-2); all these tests were negative as well. It appears that pasteurization effectively inactivates HIV, even in plasma that is likely to be highly contaminated with the virus.

Helper and suppressor T-cell function in HIV-infected hemophilia patients.

T-lymphocyte helper and suppressor functions were assessed in 61 hemophilia patients. Twenty one patients were HIV-negative (Group 1), 27 were HIV-positive without having AIDS-related complex (ARC)/AIDS (Group 2), and 13 had ARC/AIDS (Group 3). T, CD4-positive, or CD8-positive T lymphocytes were cocultured with B lymphocytes and pokeweed mitogen for 6 days and immunoglobulin producing cells were assessed in a reverse hemolytic plaque assay. In HIV-infected patients, T cells as well as the CD4-positive T cell subset exhibited reduced helper (P less than .01, Group 2; P less than .0005, Group 3) and elevated suppressor activity (P less than .02, Group 2; P less than .005, Group 3), whereas no significant difference was found between HIV-negative patients and controls. The number of CD4-positive cells was not correlated with CD4 cell function. CD4-positive cells showed no helper activity (less than 10% of control T cells) in 8/11 (73%), but an excessive suppressor activity (greater than 80% suppression of plaque formation) in 6/11 (55%) Group 3 patients. Our results show that defective helper and elevated suppressor functions of T cells in HIV-infected patients are caused not only by a change in the CD4/CD8 cell counts but also by functional abnormalities of the CD4-positive T-cell subset. These abnormal helper and suppressor functions may play a role in the development of the immunodeficiency state of AIDS patients.

Lymphocyte autoantibodies and alloantibodies in HIV-positive haemophilia patients.

Immune parameters were studied in 86 haemophilia patients (six with AIDS) and 87 healthy controls. We found lymphocytotoxic alloantibodies in HIV-positive (HIV+) sera which reacted preferentially with B lymphocytes but also with T lymphocytes, and which reacted more frequently at 4 degrees C than at 37 degrees C. The antibodies were not directed against HIV-induced structures on T lymphocytes and they were reactive with both CD4+ and CD8+ lymphocytes. In addition to cytotoxic alloantibodies, cytotoxic autoantibodies were detected which coated patient lymphocytes in vivo. Increased proportions of in vivo-antibody-coated-cells were found in 37 of 86 haemophilia patients. Antibody binding was labile so that the immunoglobulins were partially removed from the lymphocyte surface by washing. The autoreactive antibodies were of IgG and IgM type, fixed complement as demonstrated by increased anti-C3d+ cells in the patients' blood, and reacted with CD4+ as well as CD8+ lymphocytes. There was a statistically significant correlation of increased Ig+ cells with HIV infection, decreased CD4/CD8 ratios, increased serum neopterin levels, and abnormal in-vitro responses to pooled allogeneic stimulator cells or CD3 monoclonal antibody. Patients with increased Ig+ cells were lymphopenic, had decreased absolute counts of CD4+, CD25+, CD21+ and OKM5+ cells, and higher percentages of CD8+ and OKIa1+ cells in their blood than patients with normal levels of Ig+ cells. Our data suggest a role of autoreactive anti-lymphocyte antibodies in the pathogenesis of acquired immunodeficiency.

Autoantibodies against CD4- and CD8-positive T lymphocytes in HIV-infected hemophilia patients.

The presence of IgG, IgM, C3d, or gp120 on the surface of T lymphocytes was analyzed by flow cytometry in blood samples from 73 hemophilia patients and 56 healthy controls. IgG and IgM autoantibodies against CD4+ lymphocytes were found in HIV + patients but not in HIV-patients or healthy controls (p less than 0.001). IgM autoantibodies were more frequent than IgG autoantibodies. Autoantibody formation increased with disease progression. However, within the same disease risk category, patients with autoantibodies were not "more immunologically abnormal' than patients without autoantibodies. HIV + patients who possessed autoantibodies had similar CD4+ and CD8+ lymphocyte counts as HIV + patients without autoantibodies. There was no significant difference in the number of patients with abnormal CD4/CD8 ratios, serum neopterin levels, or in vitro responses to allogeneic stimulator cells or mitogens between autoantibody-positive or -negative patients of the same risk category. Our data suggest that autoantibodies against CD4+ lymphocytes may be helpful as indicators of disease progression, however, their immunopathogenetic role remains unclear.

Correlation of in vitro immune defects with impaired gamma interferon response in human-immunodeficiency-virus-infected individuals.

65 patients belonging to known acquired-immune-deficiency-syndrome (AIDS) risk groups were tested for mitogen responsiveness to pokeweed mitogen, concanavalin A and phytohemagglutinin, percentages of peripheral blood CD4, CD8 and Leu7 lymphocyte subsets, serum neopterin levels, and gamma-interferon (gamma-IFN) concentrations in cell culture supernatants. Patients with clinical symptoms of human-immunodeficiency-virus infection showed reduced gamma-IFN production in vitro. The IFN concentrations were correlated significantly with mitogen-induced blastogenesis. High serum neopterin levels and decreased CD4/CD8 ratios were associated with diminished gamma-IFN levels. The percentage of Leu7 lymphocytes was increased in a group of patients with strikingly elevated gamma-IFN levels.

On the unreliability of one-stage factor VIII:C clotting assays after infusion of factor VIII concentrates.

A multicentre study was undertaken in order to determine the reliability of the methods of assay of F VIII:C and the position of the peak value obtained after infusion of F VIII:C concentrates. Blood samples were drawn before and 10, 30 and 60 min after injection of F VIII concentrates in six haemophiliacs in one of the centres. Coded, frozen plasma samples were dispatched to the laboratories of the other four centres. F VIII:C was determined by different one-stage methods using the same international standard but with different activators. The results of the different laboratories differed widely and no agreement was reached on the existence of a lag period. To reach a valid conclusion not only the same sample has to be analysed, as in this study, but also the same laboratory technique has to be used by all participating investigators. To reach agreement on in vivo recovery and on elimination curves for different F VIII concentrates multicentre studies must be based on reliable methods of assay.

Absence of hepatitis after treatment with a pasteurized factor VIII concentrate in patients with hemophilia and no previous transfusions.

Post-transfusion hepatitis is frequent among patients with hemophilia who are treated with concentrated factor VIII prepared from pooled plasma, especially if it is obtained from paid donors. In 26 patients with hemophilia A or von Willebrand's disease who had not been treated with blood or any blood product and hence were highly susceptible to the development of post-transfusion hepatitis, we infused 32 batches of a factor VIII concentrate that had been produced from large pools of human plasma (collected from paid plasmapheresis donors) and then heated in solution at 60 degrees C for 10 hours before final lyophilization. Patients were examined clinically and serologically over a period of 12 months after the first infusion of the pasteurized concentrate. Neither hepatitis nor serologic signs of other viral infections were observed. The hemostatic effectiveness of the concentrate appeared to be satisfactory relative to untreated concentrates.