Factor VIII chromogenic assays can be used for potency labeling and postadministration monitoring of N8-GP.

UNLABELLED: Essentials Chromogenic assays may be less variable than one-stage clot assays for measuring modified factor VIII. Chromogenic assays were evaluated for N8-GP potency labeling and postadministration monitoring. There was no significant difference between chromogenic assay kits for measuring N8-GP potency. Postadministration monitoring of N8-GP was comparable to turoctocog alfa for all kits tested. SUMMARY: Background Factor VIII activity ( FVIII: C) is commonly measured using one-stage activated partial thromboplastin time (aPTT)-based clot assays. Chromogenic assays are, however, an alternative, and potency assessment in Europe is performed using chromogenic assays. One-stage clot assays are in general associated with high variability, and modified FVIII products may add to this variability. FVIII chromogenic assays may be less affected. Objectives To evaluate available chromogenic assay kits for potency labeling of polyethylene glycol-glycoconjugated turoctocog alfa (turoctocog alfa pegol [N8-GP]) and to evaluate selected chromogenic kits for postadministration monitoring of N8-GP using turoctocog alfa (Novoeight(®) ) as comparator. Methods Six FVIII chromogenic assay kits were adapted to the European Pharmacopeia guidelines for potency labeling, including assessment of time to 50% FX activation. Four kits were adapted for postadministration monitoring using an ACL(®) TOP 500 analyzer. Severe hemophilia A plasma was spiked with N8-GP or turoctocog alfa to simulate postadministration samples. The World Health Organization (WHO) 8th International Standard (IS) FVIII concentrate was used as calibrator throughout. In addition, a plasma calibrator was used for postadministration samples. Results When measuring N8-GP potency, no significant difference using a 1% significance level was observed between kits. In simulated postadministration samples, all test kits were highly accurate and precise, except at low concentrations, with no significant difference between FVIII: C (P > 0.05) measured using the different calibrators. However, values obtained using the WHO 8th IS were closer to labeled values. Conclusions Chromogenic assay kits tested measured consistent FVIII: C for N8-GP potency and showed comparable results for N8-GP and turoctocog alfa in simulated postadministration samples.

Effects of apoptosis and lipid peroxidation on T-lymphoblastoid phospholipid-dependent procoagulant activity.

BACKGROUND: Coagulation has an absolute requirement for macromolecular complexes to be assembled on a negatively charged phospholipid (PL) surface. Previously, we reported that malignant T-lymphoblastoid cells have the ability to support procoagulant activity (PCA) independently of tissue factor by providing such a surface. OBJECTIVE: To explore the effect of two pathophysiologic processes, apoptosis and lipid peroxidation (LP), on this PL-dependent PCA. METHODS: Three different assays for PL-dependent PCA (factor IXa-initiated FXa and thrombin generation and prothrombinase activity) were used to investigate this PCA after exposing three T-lymphoblastoid cell lines to either apoptotic stimuli (1 microM staurosporine) or oxidative stress (4 mm H(2)O(2) and 40 microM CuSO(4)). Surface exposure of anionic PL was measured by flow cytometry using annexin A5(FITC) and an antibody (3G4) specific for native, but not oxidized, phosphatidylserine (PS). RESULTS AND CONCLUSIONS: Both apoptosis and LP significantly enhanced the PCA of cells, to a level that was greater than that observed following calcium ionophore treatment, suggesting that the increased activity was not solely due to anionic PL exposure. Whereas cells undergoing apoptosis bound both annexin A5(FITC) and 3G4, only annexin A5(FITC) bound to cells undergoing LP. This implies that apoptosis increases PCA by causing the translocation of oxidized/native PS to the outer membrane, whereas LP appears to increase the PCA, possibly due to malondialdehyde adducts altering the net charge on the cell surface, which allows PLs other than PS to participate in thrombin generation.

Collaborative study for the establishment of replacement batches for human coagulation factor IX concentrate reference standards.

The European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) batch 1, the World Health Organisation (WHO) 3rd International Standard, Human (IS, 96/854) and the FDA Standard for human blood coagulation Factor IX concentrate have been available since 1996, following their establishment by a common collaborative study. Due to dwindling stocks of all three standards, a new WHO-EDQM-FDA tri-partite collaborative study was launched to establish replacement batches. Thirty laboratories from fourteen countries took part in the collaborative study to assign potency values to candidate preparations. Three candidates, one of recombinant and two of human plasma-derived origins, were assayed against the 3rd IS for Blood Coagulation Factor IX, Concentrate, Human (96/854). The 3rd IS for Blood Coagulation Factors II, VII, IX and X, Plasma, Human (99/826) was also included to evaluate the relationship between the factor IX plasma and concentrate unitage. Thirty-two sets of clotting assay results and two sets of chromogenic assay data were analysed. There was a significant difference in potency estimates by these two methods for the recombinant candidate (sample B) and the plasma IS (sample P). Similar potency values were obtained for the plasma derived products (monoclonal antibody- and chromatography-purified factor IX, samples C and D) by clotting and chromogenic assays. For the clotting assays, intra-laboratory variability (GCV) was found to range from 0.5 - 21.7%, with the GCV for the majority of laboratories being less than 10%. Good inter-laboratory agreement, with the majority of the GCV being less than 10% (GCV range = 4.7 - 10.6 %) was also obtained. The mean potency values estimated by the clotting assay using plasma as pre-diluent (as directed by the Ph. Eur. general chapter method) did not differ from values obtained using buffer. Taking into account the preliminary stability data, the intra- and inter-laboratory variability, and the differences between the clotting and chromogenic assay results, sample C (07/182) was established as the Human coagulation factor IX concentrate BRP batch 2, with a potency value of 7.9 IU/ampoule assigned with clotting assay results. As an outcome of this tri-partite collaborative study, the same sample C (07/182) has also been adopted as the 4th International Standard for Blood Coagulation Factor IX, Concentrate, Human by the Expert Committee on Biological Standardisation (ECBS) of the World Health Organisation (WHO), and as the replacement batch for the reference standard for Human coagulation factor IX concentrate by the FDA.

Characterization of the cell-surface procoagulant activity of T-lymphoblastoid cell lines.

The procoagulant activity (PCA) of four T-lymphoblastoid cell lines (CEM-CCRF, Jurkat, Molt-4 and A3.01) at different stages of differentiation has been characterized and compared with that of a monocytoid cell line (THP-1). Four assay systems were employed; the activated partial thromboplastin time (APTT); prothrombin time/tissue factor (TF) activity; a purified factor (F)Xa generation system and cancer procoagulant. High levels of TF activity were seen only with the monocytic cells. However the more differentiated of the T-lymphoblastoid cells (Molt-4 and A3.01) were more active than monocytic cells in supporting FXa generation. This pattern was not repeated for the APTT assay, which was related to cell-surface TF activity, since it was partially inhibited by antiTF antibody. Annexin V totally inhibited the activity observed in all three assay systems, indicating that the PCA of T-lymphoblastoid cells is primarily due to expression of negatively charged phospholipids. However, antiphosphatidylserine antibody even at a high concentration gave only partial inhibition of the activity observed in the APTT and FXa generation systems for the cells compared with almost total inhibition for the phospholipid standard, suggesting either that cellular phosphatidylserine (PS) is less accessible to the antibody, or that PS is not the sole negatively charged phospholipid responsible for this activity. Flow cytometry studies using propidium iodide and annexin V showed that the PCA, although linked to PS exposure, was not the result of apoptosis.

Anti-beta 2 glycoprotein 1 and anti-annexin V antibodies in women with recurrent miscarriage.

While it has been established that anti-phospholipid antibodies (aPL) are associated with recurrent miscarriage (RM), the importance of anti-beta2 glycoprotein 1 (GP1) IgG and anti-annexin V IgG antibodies as risk factors for RM is undefined. We have investigated the prevalence of anti-beta2 GP1 IgG and anti-annexin V IgG antibodies in 54 aPL-positive and 48 aPL-negative women with RM. The prevalence of IgG anti-beta2 GP1 antibodies was not significantly different in persistently aPL-positive women with RM (7%), aPL-negative women with RM (6%) and the normal parous control group (3%). Anti-annexin V IgG antibody prevalence was significantly increased in aPL-positive women with RM compared with aPL-negative women with RM (P = 0.01). The elevations were found in 35%, 19% and 16% of aPL-positive women with RM, aPL-negative women with RM and the control group respectively. No women showed positivity for both anti-beta2 GP1 IgG and anti-annexin V antibodies. Anti-beta2 GP1 IgG antibodies do not appear to be contributory to the investigation of women with RM. Anti-annexin V antibody positivity, although associated with aPL positivity in women with RM, is not an independent risk marker.

G20210A prothrombin gene mutation: prevalence in a recurrent miscarriage population.

Many recurrent pregnancy losses appear to have a thrombotic etiology. We have investigated the prevalence of the G20210A prothrombin gene mutation in 122 women with a history of three or more early (< or = 12 weeks gestation; n = 91), late (> 12 weeks gestation: n = 2), or mixed (n = 29) consecutive pregnancy losses. A control group of 66 healthy parous women with no history of thrombosis or miscarriage was also studied. Four heterozygotes that suffered only early pregnancy losses were detected in the patient group giving a prevalence of 3.3%. Three of the control group women were heterozygous for the mutation. giving a prevalence of 4.5% (p = 0.32: odds ratio 0.71: 95% confidence interval [CI] 0.15-3.27). When only Caucasians were analyzed, a prevalence of 3.9% (4/103) was observed in the patient group and 4.2% (2/48) in the control group (p = 0.28; odds ratio 0.89; 95% CI 0.16-5.05). The prevalence of the G20210A prothrombin gene mutation is not increased in women with recurrent miscarriage, although it was only found in women who had suffered early pregnancy losses. However, it remains possible that this mutation is relevant in a selected subgroup of women with recurrent miscarriage, additional thrombophilic defects, and in whom fetal loss is associated with placental infarction and thrombosis.

Pregnancy outcome is not affected by antiphospholipid antibody status in women referred for in vitro fertilization.

OBJECTIVE: To determine the prevalence of antiphospholipid (aPL) and anti-beta 2 glycoprotein I (anti-beta2-GPI) antibodies in women referred for IVF and to prospectively evaluate the effect of these antibodies on IVF outcome. DESIGN: Prospective observational study. SETTING: A university hospital and IVF unit. PATIENT(S): Three hundred eighty consecutive women referred for IVF. INTERVENTION(S): Blood samples taken before commencement of IVF cycles were tested for the presence of aPL (lupus anticoagulant [LA], anticardiolipin [aCL], and antiphosphatidyl serine antibodies [aPS]) and anti-beta2-GPI antibodies. MAIN OUTCOME MEASURE(S): Antibody prevalence, pregnancy rates, and live birth rates. RESULT(S): Of the total 380 women, 89 tested persistently positive for aPL (23.4%). None of 176 women tested for IgG aPS antibodies had a positive titer. Only 3.3% (11 of 329) tested positive for anti-beta2-GPI antibodies. Pregnancy rate, live birth rate, gestational age at delivery, and birth weight were not affected by aPL status. CONCLUSION(S): Although women referred for IVF have a high prevalence of aPL, these antibodies do not affect the outcome of treatment. Screening women undergoing IVF for aPL is not justified.

HEPTEST: a suitable method for monitoring heparin during pregnancy.

Methods of monitoring heparin in pregnancy are problematic. The aim of this study was to assess the plasma HEPTEST as a rapid and reliable test for heparin monitoring in pregnancy. HEPTEST, activated partial thromboplastin time (APTT) and chromogenic anti-Xa assays were performed on individual heparin-spiked plasma samples from two groups: normal non-pregnant women (n = 6) and normal pregnant women during the third trimester (n = 6). Heparin activity curves were established in plasma from both groups for low (< 0.3 IU/ml), intermediate (0.3-0.7 IU/ml) and high (> 0.7 IU/ml) heparin concentrations and validated by comparison with the anti-Xa chromogenic assay. Both the APTT and HEPTEST demonstrated good correlation with anti-Xa levels across all heparin concentrations in both plasma groups (r range = 0.879-0.945). In comparison with the APTT, the HEPTEST showed better correlation with anti-Xa levels at low concentrations of heparin (r values 0.933 vs. 0.772, respectively). For both the APTT and HEPTEST there were significant differences between the clotting times in pregnant and non-pregnant plasma at a number of heparin concentrations. This data supports the plasma HEPTEST as an acceptable alternative to the chromogenic anti-Xa assay for monitoring heparin thromboprophylaxis in pregnancy.

Acquired haemophilia A: errors in the diagnosis.

The distinction between a specific factor inactivator and a non-specific inhibitor is important when confronted by a patient with a history of bleeding and abnormal in-vitro coagulation tests. We report on two patients who presented with bleeding and a prolonged activated partial thromboplastin time. Initial factor assays suggested combined deficiency of factors VIII and IX as a result of the presence of inactivators. The use of dilution studies, chromogenic assays, a novel in-house enzyme-linked-immunosorbent-assay-based technique and phospholipid neutralization, demonstrated that Case 1 had a genuine factor VIII inactivator resulting in factor VIII levels of less than 1 IU/dl but no factor IX deficiency. Case 2 had normal levels of factor VIII on further testing and no specific inactivator to either factor VIII or IX but a potent antiphospholipid antibody which had interfered with the phospholipid-dependent in-vitro assays. Care must be taken in the interpretation of laboratory assays in the presence of antiphospholipid antibodies to ensure that the correct diagnosis is made and inappropriate treatment avoided.

Intravenous drug use and glomerular deposition of lipid-like material.

UNLABELLED: We present a series of 5 intravenous drug users (4 male, age range 24-47 years) with glomerular deposition of a lipid-like material. Three patients presented with the nephrotic syndrome, 1 with acute renal failure and 1 with hypertension and an active urinary sediment. All were continuing to use opiates intravenously (mixed with lemon juice or acetic anhydride and sodium bicarbonate). INVESTIGATIONS: plasma creatinine 0.10-0.30 mmol/l, urinary protein excretion 1.0-6.75 g/24 h, hepatitis C antibody positive (4), all seronegative for hepatitis B and HIV. Renal biopsy showed large vacuolated areas consistent with heavy deposition of lipid-like material in all glomerular cells and infiltrating macrophages. Two showed ATN and another FGS. We suggest that these changes are due to the pattern of illicit drug availability and preparation peculiar to these users.

Kindness, prescribed and natural, in medicine.

To omit the word kindness in medical practice and journals, in favour of fashionable notions such as "care" and "skills", is not in patients' interests. Health professionals may come to the view that natural kindness (the same as that found in the world outside medicine), because it is absent by name in medical skills courses' or other official edicts, is somehow unscientific and unworthy of their attention. As lay-people know, it is an essential adjunct to all medical management, sometimes the only one required, and by no means always a time-taking matter. And so its use by name in journals, and its actual use in practice, is here recommended. It is a supreme medical ally.

Antiphospholipid antibodies and beta 2-glycoprotein-I in 500 women with recurrent miscarriage: results of a comprehensive screening approach.

Five hundred consecutive women (median age 33 years; range 19-45) with a history of recurrent miscarriage (median 4; range 3-16) were screened for the presence of antiphospholipid antibodies (APA)-lupus anticoagulant (LA) and/or anticardiolipin antibodies (ACA). The prevalence of persistently positive tests for LA was 9.6% and for immunoglobulin G (IgG) and immunoglobulin M (IgM) ACA was 3.3 and 2.2% respectively. Only seven women (1.4%) were LA and ACA positive. Repeat testing, after an interval of at least 8 weeks, demonstrated that only 65.7% of LA positive, 36.6% IgG ACA positive and 36.0% IgM ACA positive women on initial testing had a second positive test result. The dilute Russell's viper venom time detected the LA significantly more often than either the activated partial thromboplastin time or the kaolin clotting time (P < 0.001). There was no difference in the gestation of previous miscarriages between APA positive and APA negative women. There was no difference in the plasma beta 2-glycoprotein-I concentrations between APA positive and APA negative women with miscarriages and normal women. All women with a history of recurrent miscarriage should be tested for the presence of both LA and ACA. A second confirmatory test should be performed in those with an initial positive test result.

Determination of antimony (III) and (V) by differential pulse anodic stripping voltammetry.

Re-evaluation of DPASV procedures for determining low levels of Sb (III) and Sb (V) in solution identified several problem areas, e.g. anomalous ASV behaviour, possible formation of an intermediate valency state during the analytical cycle, and chemical interactions in acidified test solutions containing both valency states. Specific determination of Sb (III) can be achieved using base solutions composed of 0.2M HCl (detection limit 10 nM) or acetic acid/acetate buffer (detection limit 600 nM). For the determination of Sb (V), analysis in 2M HCl is recommended [with response in 0.2M HCl being used to correct for any Sb (III) present].

The computational restoration of autoradiographic images.

A double integral formula is derived for the distortion in an autoradiographic image due to electron "cross-fire", and a deconvolution procedure for image restoration using a fast Fourier transform algorithm is developed. The technique is applied to a simulated autoradiograph of a uniform disc source and is found to give excellent results. Further image enhancement is obtained from the use of a spatially moving average smoothing process.