Co-inheritance of the 20210A allele of the prothrombin gene increases the risk of thrombosis in subjects with familial thrombophilia.

The presence of the 20210A allele of the prothrombin (PT) gene has recently been shown to be a risk factor for venous thromboembolism. This is probably mediated through increased plasma prothrombin levels. The aim of this study was to compare the prevalence of the prothrombin 20210A allele in control subjects and in subjects with recognised thrombophilia and to establish whether the additional inheritance of the PT 20210A allele is associated with an increased risk of venous thromboembolism. 101 subjects with a history of venous thromboembolism and diagnosed as having either factor V Leiden (R506Q) or heritable deficiencies of protein C, protein S or antithrombin were studied. The prevalence of the PT 20210A allele in this group was compared with the results obtained for 150 control subjects. In addition, the relationships were examined between genetic status and the number of documented thromboembolic episodes, and between plasma prothrombin levels and possession of the PT 20210A allele. 8 (7.9%) of the 101 patients were also heterozygous for the PT 20210A allele. This compares with 0.7% in the control subjects (p = 0.005). After exclusion of patients on warfarin, the mean plasma prothrombin of 113 subjects without 20210A was 1.09 U/ml, as compared with 1.32 U/ml in 8 with the allele (p = 0.0002). Among the 101 patients with either factor V Leiden, protein S deficiency, protein C deficiency or antithrombin deficiency, the age adjusted mean (SD) number of venous thromboembolic episodes at diagnosis was 3.7 (1.5) in those with the PT 20210A allele, as compared with 1.9 (1.1) in those without (p = 0.0001). We have demonstrated that the prevalence of the PT 20210A allele is significantly greater in subjects with venous thrombosis and characterised heritable thrombophilia than in normal control subjects and that the additional inheritance of PT 20210A is associated with an increased risk of venous thromboembolism. We have also confirmed that plasma prothrombin levels are significantly greater in subjects possessing the PT 20210A compared with those who do not.

Biochemical and functional characterization of mature progeny purified from a myelomonocytic leukemia cell line.

Comparative studies were performed on the cloned myelomonocytic leukemia cell line, WEHI-3B, and a subcloned line, WEHI-3BM6. WEHI-3BM6 cells were less responsive than WEHI-3B cells to differentiation factor (DF) present in the sera of mice injected with endotoxin (endotoxin serum, ES). WEHI-3BM6 cells produced only 8% monocytes after 6 days of incubation with ES compared with 68% monocytes produced by WEHI-3B cells. In the presence of ES the rate of differentiation of both cell lines was enhanced by the addition of actinomycin D (5 ng/ml) such that after 2 days of stimulation 62% of the cells were mature monocytes. Lysozyme content as well as the expression of alpha-napthyl acetate esterase were also increased by actinomycin D. As indicated by the shifts in modal fluoresence levels (209 vs 63), differentiating WEHI-3B cells showed an increase in the binding of an anti-neutrophil serum compared with untreated WEHI-3B cells. The binding of anti-neutrophil antibodies allowed the sorting of the mature monocytes from the blast cells in DF-treated WEHI-3B cells achieving a purity of 78%. Electrophoretic analysis of radiolabelled proteins from the cell extracts of WEHI-3B-derived monocytes showed close similarities to normal murine peritoneal macrophages and distinct differences from the protein profiles of purified murine peritoneal polymorphs. A protein of 75,000 mol, wt present in the polymorphs was absent from the WEHI-3B monocytes.

The platelet defect in acute myeloid leukaemia.

In a study of platelets from 13 patients with acute myeloid leukaemia abnormal aggregation and release reactions were found. A previously unrecognised quantitative defect of thromboxane B2 production may, at least in part, explain these findings. In contrast to a previous report, we were unable to show a convincing storage pool defect in these platelets. The platelet membrane glycoproteins were largely normal.

An evaluation of screening tests for defects in the protein C pathway: commercial kits lack sensitivity and specificity.

A comparative evaluation of four commercial coagulation test kits for screening the protein C pathway kits was performed at two centres. The tests were Acticlot V-OUT (V-OUT), the PCA test (PCA), the GradiThrom PCP test (PCP) and ProC Global (ProC). Reference ranges were established in 40 normal subjects and, with the exception of V-OUT and ProC, significant differences were observed between males and females. Consequently, sex-specific normal cut-off values (fifth percentile) were used that led to greatly improved sensitivity when compared with the manufacturers' recommended cut-off values. Plasma from patients with factor V Leiden (n = 23), congenital protein S deficiency (n = 19), congenital protein C deficiency (n = 11), lupus anticoagulant (n = 17) and thrombophilia with no demonstrable protein C pathway defect (n = 20) were tested. All kits showed 100% sensitivity to factor V Leiden, but sensitivity was variable for protein C deficiency (27-73%), and poor for protein S deficiency (29-35%). The lupus anticoagulant affected all kits to some degree, with 29-35% giving values below the fifth percentile of normal, whereas all kits gave 1/20 unexpected abnormal results in the thrombophilia group, with the same sample accounting for the abnormal results in three of the four kits. Overall sensitivity and specificity, respectively, for defects in the protein C pathway were: V-OUT, 60 and 91%; PCA, 70 and 86%; PCP 75 and 94%; and ProC, 66 and 88%. We conclude that all four kits lack the sensitivity and specificity required for routine laboratory screening for defects in the protein C pathway and cannot replace assays for the individual proteins of this system.

The phenotypic and genetic assessment of protein C deficiency.

This paper outlines the methods and approaches used for the laboratory detection and investigation of protein C (PC) deficiency. It does not make recommendations as to which patients should have thrombophilia testing performed; this should be done in line with local guidance. Interpretation of PC level is complicated because level varies with age, and many conditions can cause acquired deficiency. Protein C is most usually measured by chromogenic assay as a part of the thrombophilia screen. There exists, however, a very small group of individuals with significant PC deficiency, in whom the chromogenic PC assay is normal. The coagulometric assay of PC is more sensitive to these rare defects, but these assays may lack specificity. Genetic analysis allows definitive diagnosis and may be useful in confirming that deficiency is inherited and not acquired and is particularly valuable in families with severe PC deficiency.

The phenotypic and genetic assessment of antithrombin deficiency.

INTRODUCTION: Antithrombin (AT) deficiency is associated with an increased risk of deep vein thrombosis and pulmonary embolism which are major causes of morbidity and death. The incidence of deficiency in healthy populations has been reported to vary from 1/600 to 1/5000, with the variation being due to the different populations studied and detection methods used. When reduced activity levels are identified it is important to measure the AT antigen levels to differentiate type I from type II disorders, as type II defects have varying thrombotic risk. METHODS: Functional AT assays detect the ability of AT to inactivate thrombin or factor Xa, and AT antigen assays detect the quantity of AT in plasma. In functional assays, reducing the incubation time of sample with enzyme/heparin reagent may increase sensitivity to type II defects. An excess of antigen over activity level suggests the presence of functionally defective AT, which can be characterized further by assaying AT in the absence of heparin, electrophoresis to investigate the ability of heparin to bind to AT, and gene sequencing. RESULTS: Many patients with AT deficiency have a type II defect and these defects may not be detected by all routine diagnostic assays. Assays using human thrombin may lack specificity and assays that use factor Xa may fail to detect the common variant, AT Cambridge II, which can be detected by assays using bovine thrombin, especially if activity is compared to antigen by ratio. Factor Xa based assays may be particularly sensitive to certain heparin binding defects, and sensitivity of assays to both heparin binding and reactive site defects can be improved by shortening the incubation time with enzyme. CONCLUSION: uAT activity assays are essential for the detection of AT deficiency because type II defects are relatively common in patients with heritable deficiency. No one functional assay can be assumed to detect all forms of AT deficiency, and assays can sometimes be improved by reducing reaction time of AT with thrombin or factor Xa.

Marked discrepancy between coagulometric protein C activity assays with the pro-thrombotic protein C Asn2Ile substitution.

INTRODUCTION: Protein C (PC) Asn2Ile is a rare pro-thrombotic variant in which loss of anticoagulant function in vivo is likely to result from defective PC binding to the endothelial PC receptor and phospholipid. METHODS: To characterize the consequences of this functional defect on the diagnostic performance of commercial PC activity assays, we performed one antigen, three chromogenic and three coagulometric assays on plasma from a subject who was heterozygous for the PC Asn2Ile substitution. RESULTS: As anticipated, the PC antigen (96.2 IU/dl) and chromogenic PC activity assays (98.4 IU/dl) were insensitive to PC Asn2Ile, which has intact enzymatic activity and is secreted normally. There was a marked reduction in apparent PC activity by STACLOT coagulometric assay (50.4 IU/dl). However, there was only a small reduction in apparent PC activity by CRYOcheck Clot C assay (69.5 IU/dl) and by HemosIL ProClot assay, the PC activity was within the laboratory reference range (91 IU/dl). This discrepancy between coagulometric assays could not be explained by lupus anticoagulant, activated PC resistance or elevated plasma factor VIII activity. CONCLUSION: We demonstrate that coagulometric assays are not equally sensitive to clinically important functional defects of PC and that multiple assays may be required to identify all variants.

Clinical multicenter evaluation of a new FXa-based Antithrombin assay.

INTRODUCTION: The determination of functional Antithrombin is a central part of thrombophilia screening. In this multicenter study, a new FXa-based method (INNOVANCE® Antithrombin) was evaluated on four different analyzers. METHODS: The INNOVANCE Antithrombin method was evaluated by precision and reference interval studies and by comparing the new method with established methods through parallel measurement of samples from 249 patients and 151 apparently healthy individuals. RESULTS: The INNOVANCE Antithrombin assay demonstrated on all analyzers repeatability coefficients of variation (CVs) ≤ 3.2% and within-device and between-run CVs ≤ 6.9%. The reference intervals of all analyzers are comparable with 2.5th percentiles between 80% and 85% of normal. The INNOVANCE Antithrombin and the FIIa-based Berichrom® AT III (A) methods demonstrated good concordance with correlation coefficients of r = 0.908 or higher. The INNOVANCE Antithrombin method demonstrated furthermore an excellent comparability with the STA® Antithrombin III assay and an acceptable comparability with the Coamatic® LR Antithrombin assay. The patients with congenital deficiency (n = 31) were identified with all assays except for the patients carrying the P41L heparin-binding site mutation, which was only identified with the INNOVANCE Antithrombin and the STA Antithrombin III methods. CONCLUSION: The INNOVANCE Antithrombin assay has high sensitivity for Antithrombin deficiencies and is reliable, precise and suitable for routine clinical use.

The effect of tissue factor concentration on calibrated automated thrombography in the presence of inhibitor bypass agents.

Thrombin generation has been suggested as a method to monitor treatment with factor eight inhibitor bypassing activity (FEIBA) or recombinant FVIIa (rFVIIa). The sensitivity of the assay for individual coagulation factors is dependent on the tissue factor (TF) concentration. An inverse relation between the rFVIIa concentration needed to shorten the clotting time and TF concentration has been shown but the data on thrombin generation are inconsistent. Information on TF concentration in measurements with FEIBA is limited. We studied the influence of TF concentration (1 and 5 pM) on thrombin generation through spiking experiments with rFVIIa and/or FEIBA in the plasma of severe haemophilia A patients and after four and three treatment episodes, respectively, using the calibrated automated thrombin generation assay (CAT) in platelet poor plasma. Spiking with FEIBA showed a linear relation with the endogenous thrombin potential (ETP)/peak at 1 pM but substrate depletion at 5 pM. Spiking with rFVIIa showed a near linear dose-response relation with the ETP/peak at 1 pm but only a shortening of the initiation phase at 5 pM. Similar effects were present in post-treatment samples. FEIBA acted synergistically with rFVIIa. This suggest a role for CAT in monitoring inhibitor bypass treatment but low TF concentrations are required to avoid substrate depletion with FEIBA and to demonstrate the effect of rFVIIa.

Evaluation of a new venom-based clotting assay of protein C.

Congenital protein C deficiency significantly increases the risk of venous thromboembolism, a serious and potentially lethal condition. Protein C levels can be determined by chromogenic, clotting and antigenic assays, each type of assay has differences in specificity and sensitivity to protein C deficiency. In principle, clotting-based assays of protein C are preferred over chromogenic assays, as they can detect some rare mutations that are missed by the chromogenic assay, however, clotting-based assays may be prone to inaccuracy because of poor specificity. We have evaluated a new venom-based clotting assay of protein C, and optimized it for use on Sysmex CA-1500 analyser. The assay was linear from 0 to 130 U/dl, a normal plasma demonstrated good inter-assay precision, with a coefficient of variation of 4.8%. The assay compared well with antigenic- and venom-based chromogenic protein C assay in normal individuals, subjects with lupus anticoagulant, and subjects with FV Leiden. Median protein C levels by clotting, chromogenic and antigen for the three subject groups were 108 U/dl, 108 IU/dl and 109 IU/dl for normal subjects, 94 U/dl, 106 IU/dl and 103 IU/dl for subjects with lupus anticoagulant, and 102 U/dl, 104 IU/dl and 100 IU/dl for subjects heterozygous for FV Leiden. Comparing levels of clotting protein C with protein C antigen by ratio (clotting/antigen), the three groups showed small differences that did not quite reach statistical significance, (mean ratios ranged from 0.95 to 1.01, anovaP = 0.0561), the lowest ratio was with the lupus anticoagulant group. Comparing clotting assay with chromogenic assay by ratio (clotting/chromogenic), the three groups did show a statistically significant difference (P = 0.0033) which was due to a difference in mean ratios between normal and lupus anticoagulant groups (ratios 1.00 and 0.91, respectively, P < 0.01). There was no statistical difference in any of the groups when comparing chromogenic protein C with protein C antigen (mean ratios ranged from 1.02 to 1.05, P = 0.3925). In a normal sample, the clotting-based protein C level was unaffected by increasing FVIII level by up to 1000 IU/dl, using intermediate purity FVIII concentrate. The new assay is considered to be a suitable assay for the routine diagnosis of protein C deficiency.

An overview of methods for detection of factor V Leiden and the prothrombin G20210A mutations.

Venous thromboembolism, represented by deep venous thrombosis and pulmonary embolism, is a common disease with high mortality and morbidity. Within the last 25 years, risk factors for venous thromboembolism have been linked to mutations in the genes of the coagulation/anticoagulation system. Factor V Leiden and the prothrombin G20210A mutations are the most prevalent inherited risk factors predisposing to venous thromboembolism in the Western world. Tests to detect these mutations are carried out when investigating a personal or family history of venous thromboembolism. At the present, there are several different methods available for the detection of these mutations in the laboratory. The choice of the method will depend on many variables. This article is aimed at reviewing the available methods for the detection of factor V Leiden and prothrombin G20210A mutations, their principle, applicability, advantages and disadvantages of use.

The gap between: being and knowing in Zen Buddhism and psychoanalysis.

The author discusses various relationships derived from the image of gap, precipice, and abyss with specific emphasis on interacting dynamics between being and knowing as explicated in the Zen Buddhist teachings of Hui-neng and in the psychoanalytic writings of Wilfred Bion. While of significant value to psychoanalysis, it is argued that symbolic meanings can occlude the actuality of the analysand's or of the spiritual seeker's affective experiencing, particularly concerning the human tendency to concretize experiential states engendered through meditation and/or the psychoanalytic encounter. The author draws from Matte-Blanco's explication of symmetrical and asymmetrical perceptual modalities to discuss the fluid nature of spiritual experiencing, paradoxical coexistence of ultimate and relative realities and reciprocal dynamics and identities between states of experiencing that might otherwise appear opposed. The primacy of experiencing for both disciplines, particularly concerning the experiencing subject's momentary state of consciousness, forms a central theme for both Zen and psychoanalysis. Brief clinical vignettes support and illuminate the author's points.

Two new E colicins, E8 and E9, produced by a strain of Escherichia coli.

We have isolated a strain of Escherichia coli from chicken caeca which produces two E colicins and colicin M. This strain has seven plasmids, five of which have been transferred to E. coli K12. Two E. coli K12 derivatives which produce the two E colicins separately have been tested against seven standard E colicin producing strains which define seven different immunity groups. Our results indicate that these new E colicins define two further immunity groups, E8 and E9.

Electrophoretic analysis of the cytoplasmic and nuclear protein changes after induction of differentiation in WEHI-3B myelomonocytic leukemia cells.

In response to a differentiation factor (G-CSF) the myelomonocytic leukemia cell line (WEHI-3B(D+) differentiates to form mature macrophages and neutrophils. The effect of G-CSF on WEHI-3B(D+) differentiation was augmented by low concentrations (5 ng/ml) of actinomycin D. Quantitative binding of an antineutrophil serum was used to segregate the differentiated cells from the leukemic blast cells. Molecular markers of later myeloid differentiation were detected in myelocytes and macrophages purified from differentiating WEHI-3B(D+) cells. To study the initial molecular processes associated with the initiation of WEHI-3B(D+) cells to differentiation, the protein changes were analyzed using gel electrophoresis. Quantitative analysis of the fluorographs from the two-dimensional (2D) electrophorograms of the 35S-labeled proteins revealed major changes in the biosynthetic rates for 16 proteins within 5 hr: The biosynthesis of six proteins was increased and another ten proteins were synthesized at a reduced rate. Two of the proteins (17K and 36K daltons) were located in the nucleus. Pulse-chase experiments indicated that protein turnover for these proteins was rapid but the degradation of four proteins was suppressed. At least six of the proteins (16K to 120K daltons) were acidic and were associated with the cytoplasm. Electrophoretic analysis of the 35S-labeled proteins indicated that a 35K protein induced by G-CSF was found in high abundance only in purified cells of intermediate differentiation (eg, myelocytes). Other proteins (eg, a very high molecular weight protein, and a 16K dalton protein) were obviously late markers of differentiated neutrophils or macrophages.

Three immunity types of klebicins which use the cloacin DF13 receptor of Klebsiella pneumoniae.

We have investigated a group of bacteriocinogenic strains used in the epidemiological investigation of Klebsiella infections. Transfer of plasmids from these strains to laboratory strains allowed the identification of three klebicins which use the cloacin DF13 receptor in Klebsiella, but are of three distinct immunity types. These klebicins use the ferric-aerobactin receptor determined by ColV-K30 in Escherichia coli, which is also used by cloacin DF13. We propose to call them group A klebicins, of immunity types A1, A2 and A3. On the basis of immunity, cloacin DF13 also belongs to the klebicin A1 group.

Biosynthetic labeling with 32P: radiation damage to mammalian cells.

Theoretical calculations showed that biosynthetic radiolabeling of cells using typical concentrations of 32P (1 mCi/ml) resulted in high radiation doses (200-500 rad/h) being absorbed by the cells. Subsequent investigations with a mouse myelomonocytic leukemia cell line (WEHI-3B(D+)) showed significant loss of replicative ability during brief (less than 1 h) exposures to 1 mCi/ml of 32P. Complete loss of cell replicative ability was found with isotopic doses less than 100 rad (i.e., 100 muCi/ml for 5 h). Experiments employing a less radiosensitive pre-B-cell line (18.81) revealed that significant loss of viability occurred during incubation with 32P under identical conditions to those employed for the WEHI-3B(D+) cell line. Control experiments utilizing decayed batches of 32P and physical separation of the isotope solution from the cells confirmed that the cytotoxicity was caused by radiation emission rather than the presence of toxic components in the isotopic solution. The radiation doses absorbed by cells biosynthetically labeled with 59Fe, 33P, 35S, and 14C were calculated. Although significant levels of radiation can be absorbed 32P was considerably more radiotoxic than the other isotopes. The results of calculations indicated that the judicious choice of container geometry could reduce the absorbed radiation dose from 32P solutions. In particular the biosynthetic radiolabeling of cells in capillary tubes (diameter less than 1 mm) can reduce the absorbed rate to less than one-tenth of the dose received by cells suspended in Petri dishes or centrifuge tubes.

Incompatibility between E colicin plasmids.

We have tested the ability of pairs of colicin E plasmids to replicate stably in the same cell line. Although many of the pairs of E colicin plasmids were compatible, plasmids ColE3-CA38, ColE7-K317 and ColE8-J were mutually incompatible, as were ColE5-099, ColE6-CT14 and ColE9-J. Incompatibility between ColE6-CT14 and ColE5-099 or ColE9-J was asymmetrical, whereas incompatibility between the other plasmid pairs was symmetrical.