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.

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.

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.

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.

[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.

[The effect of tri-iodinated contrast media on clotting. An investigation of thrombocyte aggregation (author's transl)]. / Einfluss trijodierter Röntgenkontrastmittel auf die Blutgerinnung. Untersuchung der Thrombozytenaggregation.

In vitro investigations showed that renographic and cholecystographic contrast media reduced thrombocyte aggregation proportionate to contrast concentration. In vivo this disturbance of thrombocyte function could not be demonstrated. Other factors investigated, such as clotting time and thrombocyte counts, remained unaffected in vivo and in vitro. Age and sex and the presence of various types of pathology were also found to have no affect. These findings and their significance are discussed.

[Ultrasound diagnosis of muscle haematomas in haemophiliac patients (author's transl)]. / Ultraschalldiagnostik von Muskelhämatomen bei Hä mophiliepatienten.

Ultrasound was used for the diagnosis of muscle haematomas in seventeen patients with congenital clotting abnormalities. Fifteen patients had haematomas in the extremities or the gluteal muscles, whereas in four cases there was retroperitoneal bleeding. In three of the nineteen examinations there was no sonographic evidence of recent bleeding at the first attempt, but in sixteen cases the size, localisation and relationship to neighbouring organs could be demonstrated. It was possible to distinguish between localised haematomas and diffuse ones. Comparison of the two sides was used with semi-quantitative evaluation of the amplitude profile. The condition in the extremities favours ultrasound examination and makes it possible to use high frequencies in order to achieve better resolution; the diagnosis of bleeding into the retroperitoneal space may be difficult. At times other methods of examination, particularly computer tomography, may have to be used.

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.

[Post-transfusion hepatitis and its sequelae in the treatment of hemophilia]. / Die Posttransfusionshepatitis (PTH) und ihre Folgen bei der Hämophilie-behandlung.

As a result of the frequent application of factor VIII and IX concentrates of single donor cryoprecipitates as well as of concentrates from large plasma pools a very important side-effect became evident: the transmission of serum hepatitis in its two forms B and Non A Non B. Dependent on the factor dosage, up to 100% of the patients showed signs of an active hepatitis or contact of the defense system with hepatitis viruses. With increasing frequency of chronic hepatitis (65%) there is also an increase in the aggravation to liver cirrhosis which manifests itself 13 years earlier than in the normal population. In future, greatest attention will have to be devoted to avoiding the transmission of hepatitis and other viral infections by concentrates of clotting factors.

[A controlled study of long-term treatment of haemophilia B on an out-patient basis (author's transl)]. / Die ambulante Dauerbehandlung der Hämophilie B. Eine kontrollierte Studie

Eight patients with severe or moderately severe haemophilia B were treated for six months, according to three schemata for two months each: 18, twice 18, or twice 9 U of factor IX per kg body-weight weekly. The sequence of the six possible treatment schemes was determined strictly at random. One patient had to be excluded because he developed hepatitis, another because allergic signs developed. In the pre-trial period the number of bleedings per two months had been about 40, as recorded by the patients. The bleedings were reduced to nine after 18 U of factor IX per kg body-weight weekly, falling to two after twice 9 and twice 18 U/kg weekly. The patients themselves considered twice 9 U/kg as the ideal dosage. It is recommended that this dosage scheme should be used initially if one decides to employ permanent substitution in haemophilia B. Once freedom from bleedings and strengthening of the motor system have been achieved one can then try 18 U per kg body-weight once a week.

Selected blood coagulation parameters during extracorporeal circulation.

We investigated selected coagulation parameters in 13 patients who had undergone open heart surgery with extracorporeal circulation (ECC). During ECC factor XIIa increased considerably to 38%. This may lead to intravascular coagulation, which is however inhibited during ECC by the administered heparin. The C1-inhibitor activity decreased to 12% during ECC, this decrease was considerably greater and lasted longer than the decrease of the C1-inhibitor concentration, which declined to 59%. The levels of the other coagulation factors I, II, V, VII, X, XII, antithrombin III, alpha 2-antiplasmin and platelets decreased to 50-60%. The latter was predominantly due to hemodilution at the beginning of ECC. We assume that the critical period for the coagulation system in ECC appears when ECC is discontinued. At this point we have a high factor XIIa level and a low C1-inhibitor activity so that intravascular coagulation (DIC) may occur because then the inhibitory effect of heparin is neutralized by protamine.

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.

[A controlled study of treating haemophilia A on an out-patient basis (author's transl)]. / Die ambulante Dauerbehadnlung der Hämophilie A. Eine kontrollierte Studie

For six months 36 U factor VIII concentrates per kg bodyweight and week were administered to six out-patients with severe haemophilia A. The injection regimen was changed in every patient every two months, from 36 U/kg once to 18 U/kg twice and 12 U/kg three times, intravenously. The six possible combinations of these three dosage schedules were used in the patients in a strictly randomised manner, and all patients were treated during the same period. In the pre-trial period (treatment as needed) there were an average of 35 bleedings per two months. On continual treatment there were 21 bleedings on weekly injections of 36 U factor VIII per kg, 14 on twice weekly 18 U/kg and none on 12 U/kg, three times weekly. The differences are statistically significant. The absence of bleeding on the last dosage schedule was achieved during normal working. Days lost from work per patient per month was zero on three times 12 U/kg, 0.4 day on twice 18 U/kg and once 36 U/kg, while it had been five days in the pre-trial period. In addition to freedom from bleeding and no lost days from work, there was increased mobility and physical capacity.

[Treatment of haemophilia with factor VIII inhibitor by giving isoagglutinin-free factor VIII concentrate (author's transl)]. / Therapie von Hemmkörperhämophilie mit isoagglutininfreiem Faktor-VIII-Konzentrat

Two nine-year-old boys with haemophilia A and circulating inhibitor against factor VIII were given large doses of commercial AHG concentrates to control bleeding. But the isoagglutinins in the AHG concentrates caused haemolysis in both patients. When AHF (Immuno) from AB donors - a cryoprecipate without isoagglutinins - was used no further inhibitor activity was detectable, previously observed anaphylactoid reactions no longer occurred and usual doses of AHG were now sufficient to control bleeding.

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.

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.