One-stage Assay Research Articles

An improved method for the evaluation of coagulation times

The reciprocal relation between coagulation time t and concentration S of coagulation factors (in one-stage assays substrates in the reactions involved) can be written as t = t min + e 1 S . Here t min is the minimum possible coagulation time and e the sensitivity of the method to a change in the concentration of substrate. As the variance of coagulation time strongly increases with increasing dilution of the coagulation factors in calibration this equation should be multiplied by S for harmonizing the variance: t · S = e + t min · S . The plot of t · S against S (parametrizing straight line) now gives statistically controllable numerical values for e and t min. Deviations at low concentrations that are associated with the diluent used and caused by lack of fibrinogen, change of the pH, glass activation, or the residual content of adsorbed plasma, can be easily recognized and in most cases compensated. The minimum number of individual plasmas required for calibration can be given as well as the optimum spacing of the plasma concentrations. With e and t min known a nomogram can be constructed relating coagulation time to the content of coagulation factors in the patients' plasmas. The content can also be determined by recording a parametrizing straight line on the patient's plasma - a variant to be preferred for contents near to normal and if inhibitors are to be expected. A comparison of several commercial thromboplastin time reagents as to their sensitivity was made and the standardization of the British Comparative Thromboplastin re-examined. Single factor sensitivities can be computed and the usability of coagulometers can be considered. As the proposed method controls procedures, reagents, and coagulometers an international standardization of coagulation time measurement on its basis is thought possible.

Standardization of the one-stage assay for factor VIII (antihemophilic factor).

Although considerable progress has been made in perfecting the one-stage assay for factor VIII (antihemophilic factor), there remain variables that influence test results within laboratories as well as reproducibility between laboratories which have not been adequately evaluated. The purpose of this paper is to elucidate certain aspects of this assay that have not received adequate consideration and to describe the authors' assay method in order to provide a basis for comparison with results from other laboratories. It appears that variability results from: (1) differences in coagulability of different batches of substrate plasma obtained at different times from the same individual; (2) instability of some batches of stored substrate or standard plasmas; (3) variation in coagulability among vials of stored substrate or standard plasma from the same batch; (4) variation due to non-plasma reagents and instrumentation used to execute the test.

Altered Factor VIII Complexes in Patients with Acute Respiratory Insufficiency

Acute respiratory failure is an often-fatal syndrome of multiple etiologies in which altered factor VIII may be a marker of endothelial disease. 12 women with overwhelming viral pneumonia were studied with serial factor VIII antigen, procoagulant activity, and von Willebrand's factor assays. Antigen levels were elevated (range: 86--1644%) out of proportion to procoagulant activity (range: 35--521% by a one-stage assay), and factor VIII antigen to activity ratios were as high as 16:1. Von Willebrand's factor was normal but correlated best with procoagulant activity. All patients had abnormal antigen patterns on crossed immunoelectrophoresis, with increases in protein of both fast and slow mobility. These changes in factor VIII correlated with the patient's clinical courses.

Some sources of error in the one-stage assay of factor VIII.

The error of the one-stage assay of factor VIII activity is determined by the quality of reference and substrate (factor VIII-deficient) plasmas, as well as by the accuracy of dilution and clot observation time. Storage of the fresh normal plasma used as reference and/or of the substrate plasma for 2--3 h at 0 degrees C may cause considerable errors, which can be eliminated by the immediate use of plasma or by the determination of an appropriate correction factor. With all precautions taken into account the average error of the assay in our hands is 20%.

An International Collaborative Assay of Factor VIII Clotting Activity

SummaryAn International Collaborative Study was organised to replace the first International Standard for factor VIII. A freeze-dried concentrate, 73/552, and a freeze-dried plasma, 75/510, were assayed against the International Standard, and also compared to fresh normal plasma and local standards.In assays of the concentrate 73/552 against the first I.S. the mean potency was 1.14 i.u./ ampoule and there was no significant difference between one-stage and two-stage methods. When assayed against average fresh normal plasma, the potency was 1.05 “normal plasma units” per ampoule. It was agreed by the participants that the potency of 73/552 be regarded as the mean of these two figures, i.e. 1.10 i. u./ampoule.In assays of the freeze-dried plasma, 75/510, against the first I.S. the mean potency was 0. 68 i. u./ampoule, but the one-stage assays gave significantly higher potencies (mean 0.74 1. u./ampoule) than the two-stage assays (mean 0.59 i. u./ampoule). The same trend was also seen in the fresh normal plasmas, and in the local plasma standards. This finding has important implications for the standardisation of factor VIII.Stability studies on the concentrate 73/552 gave a predicted loss of 0.02% per year at – 20° C. All participants agreed that the material was suitable to serve as an International Standard, and at the 26th meeting of the Expert Committee on Biological Standardisation of the World Health Organization, the material in ampoules coded 73/552 was established as the 2nd International Standard for factor VIII, with a potency of 1.10 i. u./ampoule.

Factor V in an industrial population.

Factor-V levels have been measured in a random sample of 626 men and 307 women working in a variety of occupations in North West London. The method is an automated one-stage assay using the same batch of freeze-dried thromboplastin, all results being expressed in terms of the same freeze-dried standard plasma; it has been shown that only one dilution of test plasma is necessary. Factor-V levels are significantly higher when venepuncture is difficult than when it is satisfactory, the mean levels being about 130% and 117% respectively. Factor-V levels are approximately normally distributed; they are similar in men and women and in blacks and whites, and increase significantly with age at the rate of about 0.6% per annum. Factor-V levels are not affected by oral contraceptives or the menopause, and there are no differences according to blood group or secretor status.

Carrier detection in classic hemophilia by combined measurement of immunologic (VIII AGN) and procoagulant (VIII AHF) activities.

Accurate carrier detection in classic hemophilia has been difficult because of (1) technical problems related to the performance of both immunologic (VIII AGN) and procoagulant (VIII AHF) determinations, and (2) statistical problems related to the analysis of these data. VIII AHF was determined by a one-stage assay based on the partial thromboplastin time (PTT). VIII AGN was measured by the method of quantitative immunoelectrophoresis. The discriminant function U. = 0.67 1n (AHF) -- 3.17 AGN X 10(-3) was calculated for a validation group of 20 normal persons and for seven obligate carriers, and tested for accuracy of prediction on a cross-validation group of seven additional normal women and ten additional obligate carriers. A U. score of greater than or equal to 2.54 correctly identified 25 of 27 normal persons. Sixteen of 17 obligate carriers had U. scores below 2.54. In addition, of seven possilbe carriers, four were identified as normal and three as carriers. Five normal women taking oral contraceptives had disproportionately high U. scores. It is concluded that detection of carriers of classic hemophilia should be possible in the clinical laboratory by calculation of a discriminant function from combined measurements of VIII AGN and VIII AHF.

Viper venoms and coumarin-induced prothrombin. A comparison of several one-stage methods employing three different venoms as thromboplastins.

Three venoms obtained from three vipers, namely Echis carinatus, Notechis scutatus scutatus and Oxyuranus scutellatus, have been used as thromboplastin in a one-stage assay of coumarin-induced prothrombin. Regardless of the venom used, prothrombin resulted to be low in coumarin-treated patients. The mean values obtained were 27.2, 33.6, and 24.2%, respectively. These values were comparable to those obtained by means of the classical one-stage method (24.8%). A good correlation was observed among the different methods. However, the levels observed using the Notechis scutatus scutatus venom method were slightly higher as compared to those obtained by means of the other viper venoms and by means of the classical one-stage method. The three viper venoms used seem unable to activate coumarin-induced prothrombin. The levels obtained were in fact, in each instance, definitely lower than those observed immunologically. Methods which employ these viper venoms may be used in the evaluation of prothrombin in coumarin-treated patients.

The Effects of Hepes Buffer on Clotting Tests, Assay of Factors V and VIII and on the Hydrolysis of Esters by Thrombin and Thrombokinase

Shorter clotting times were found in the presence of 50 mM Hepes (N-2-hydroxyethylpiperazine-N1-2-ethanesulfonic acid) buffer than of 50mM Imidazole buffer in one-stage assays of factors V and VIII, in modified APTT and PT tests and in tests of the clotting of human plasma by purified human thrombin. All tests were performed at ionic strength 0.155 in the presence of either Hepes. NaOH or Imidazole. HCl buffer, pH 7.4 at 37 degrees. The faster clotting in the presence of Hepes buffer, therefore, is probably due, at least in part, to acceleration by Hepes of thrombin's enzymatic action on fibrinogen and/or of the polymerization of the fibrin monomers. Hepes may also have effects of other blood clotting reactions. Rates of hydrolysis of TAME or BAME (p-toluenesulfonyl- or benzoyl-L-arginine methyl ester) at pH 7.4 37 degrees by purified human bovine thrombin were essentially the same in 200 mM Hepes as in 250 mM Tris. HCl buffer (rates in Hepes. NaOH or Hepes. KOH buffers were compared with those in Tris. HCl plus NaCl for KCl). However, with purified bovine thrombokinase, rates of TAME hydrolysis in Hepes buffer were accelerated and rates of BAME hydrolysis slightly inhibited. Hepes, therefore, reacts with thrombokinase but whether this accelerates (or inhibits) the rate of converting prothrombin to thrombin remains to be determined. In addition, Hepes has an inhibitory effect on clotting since increasing the concentration of Hepes from 50 mM to 200 mM inhibits clotting in the PT, APTT and bovine thrombin-human plasma tests. Hepes buffer is being added to some plasmas and to some reagents used in clotting tests. It is, therefore, important to realize that its concentration must be monitored closely or erroneous results may be obtained in clotting tests and assays of clotting factors. The clotting times were the same in the presence of 50 mM Tris. HCl as in Imidazole. HCl buffers in APTT tests at three ionic strengths but they differed slightly in plasma-thrombin tests. Depending upon the ionic strength, 17 mM Barbital Sodium. HCl buffer inhibited APTT tests but accelerated plasma-thrombin tests. All the buffers tested, therefore, have individual effects on the clotting tests.

Sodium Periodate Modification of Factor VIII Procoagulant Activity

Chemical treatment of human factor VIII by sodium periodate resulted in increased levels of procoagulant activity when measured by the two-stage assay. Increased levels of factor-VIII activity were observed following periodate treatment of factor-VII concentrates and normal, haemophilic and von Willebrand's disease plasmas. However, sodium periodate treatment of haemophilic plasmas which were completely deficient in factor-VIII activity did not cause any reduction in the clotting times of two-stage assays. No increase in procoagulant activity following oxidation of factor VIII could be detected by the one-stage assay method. Gel filtration of factor-VIII concentrates before periodate treatment showed that the protein exhibiting factor-VIII activity had an apparent molecular weight in excess of 1 000 000, whilst after periodate treatment the apparent molecular weight was reduced to 690 000.

Reduced tissue factor (thromboplastin) activity in von Willebrand's disease

Identification of tissue factor in cultures of human skin fibroblasts permitted assessment of tissue factor activity in patients with bleeding disorders. Tissue factor activity was studied by means of a one-stage assay patterned after the prothrombin time and a two-stage test in which activation of factor X by tissue factor in the presence of factor VII was assessed. Tissue factor activity observed in cultured skin fibroblasts from five subjects with hemophilia A and B was not significantly different from that observed in pooled fibroblasts from normal controls. By contrast, tissue factor activity in cultured skin fibroblasts from four subjects with von Willebrand's disease was significantly lower than that in the control. It is postulated that the observed tissue factor defect may be related to the pathogenesis of von Willebrand's disease.

Reliability of Laboratory Tests for the Control of Oral Anticoagulation

SummaryThe present study concerned the reproducibility of the so-called prothrombin time as assessed with a series of more commonly used modifications of the Quick’s onestage assay procedure, i.e. the British comparative reagent, homemade human brain thromboplastin, Simplastin, Simplastin A, and Thrombotest. All five procedures were tested manually on pooled lyophilized normal and patients’ plasmas. In addition, Simplastin A and Thrombotest were investigated semiautomatically on individual freshly prepared patients’ plasmas. From the results obtained, the following conclusions may be drawn :The reproducibility of results obtained with manual reading on lyophilized plasmas is satisfactory for all five test procedures. For Simplastin, the reproducibility of values in the range of insufficient anticoagulation is relatively low due to the low discrimination power of the test procedure in the near-normal range (so-called low sensitivity of rabbit brain thromboplastins). The reproducibility of Thrombotest excels as a consequence of its particularly easily discerned coagulation endpoint.The reproducibility of Thrombotest, when tested on freshly prepared plasmas using Schnitger’s semiautomatic coagulometer (a fibrinometer-liJce apparatus), is no longer superior to that of Simplastin A.The constant of proportionality between the coagulation times formed with Simplastin A and Thrombotest was estimated at 0.64.Reconstituted Thrombotest is stable for 24 hours when stored at 4° C, whereas reconstituted Simplastin A is not.The Simplastin A method and Thrombotest seem to be equally sensitive to “activation” of blood coagulation upon storage.

Studies on the stability of Bovine plasma factor V

(NH 4) 2SO 4 precipitation and cellulose phosphate chromatography, commonly used to prepare plasma Factor V, have been found to produce degradation products of lower molecular weight and higher specific activity than the native protein. The so-called forms L and C ( G. Philip, J. Moran and R. W. Colman, Biochemistry, 9 (1970) 2212) were shown not to be constituents of native Factor V but instead artifacts of preparative procedures. The inclusion of Ca 2+ in the eluting buffer during gel filtration of bovine plasma Factor V allowed retention of most of the biological activity generally lost by this procedure. Concentration of Ca 2+ higher than 0.001 M depressed the activity of factor V as measured in the one-stage assay.

Clinical application of a new assay of factor VIII and factor VIII inhibitors

A new factor VIII assay with use of freeze-dried, standardized reagents was applied for investigation of healthy subjects as well as hemophiliacs, carriers and patients with von Willebrand's disease. Factor VIII activity showed no differences between men and women but a highly significant increase with age in both sexes. The new factor VIII assay showed a fairly good correlation with a well documented one-stage assay in patients with decreased factor VIII activity. A technique for quantitation of factor VIII inhibitors using the present factor VIII assay is presented. The effect of treatment with factor VIII concentrate in a severe hemophiliac with an acquired factor VIII inhibitor was studied. The calculated effect of in vitro determined inhibitor activity was in accordance with in vivo findings. The new factor VIII assay was found to be a quickly performed, sensitive method which was less susceptible than a one-stage method to the disturbing influence of traces of thromboplastin in the plasma samples.

Procoagulant synthesis by cultured fibroblasts triggered by cell adhesion.

SYNTHESIS of a potent procoagulant by cells in tissue culture has been demonstrated but its identity is obscure1. Activity is low at the beginning of culture of normal human fibroblasts and increases several hundred-fold after 12–24 h of incubation at 37° C. This increase is readily detected by the one-stage assay for factor VIII (antihaemophilic globulin) but can also be demonstrated by the two-stage factor VIII assay based on the thromboplastin generation test. Studies in progress to characterize this procoagulant have demonstrated a marked increase of factor XI as well as factor VIII activity and evolution of factor XI activity during contact activation with celite. Increase in activity detectable in the one-stage assay for factor IX and XII was much less. The procoagulant was incapable of clotting a fibrinogen solution and no change in assays for factor V and VII was observed during culture.

A Method to Study Inhibitors of Plasminogen Activation

SummaryA method to study inhibitors of plasminogen activation is described. It is mainly thought to be a one-stage plasminogenolytic method. The same type of inhibition is found in this one-stage assay system and in a two-stage caseinolytic assay system when epsilon aminocaproic acid and salicylic acid derivatives are investigated in parallel in both assay systems. Epsilon aminocaproic acid and salicylic acid derivatives inhibit the activation of plasminogen by urokinase in a competitive way. Alpha2-macroglobulin and antithrombin III did not inhibit the activation of plasminogen by urokinase.

Preparation of canine factor VII deficient substrate plasma.

Abstract An essential reagent for the specific one-stage assay of clotting factor VII (proconvertin) is substrate plasma containing an adequate complement of fibrinogen, factor II (prothrombin), factor V (proaccelerin), and factor X (Stuart) but deficient in factor VII. In the past, such plasma was available only from patients homozygously deficient in factor VII, and frequently was in scarce supply. A method is described in which mongrel dogs are given a large single intravenous injection of warfarin. Because canine plasma has a high level of factor X, a point in time following warfarin administration may be selected at which the plasma factor VII level has fallen to undetectable levels while the plasma factor X level is still over 40 per cent of normal (human). A variety of plasmas were simultaneously assayed for factor VII with canine and human factor VII deficient substrate plasmas. The results of these comparisons indicate that the use of canine factor VII deficient substrate plasma provides a reliable method for assaying factor VII.

Assay of Blood Coagulation Factors in Heparinized Blood*

SummaryTechnics have been devised permitting assay of certain coagulation factors in blood despite the presence of heparin in the sample. In the process of developing these methods, heparin and Polybrene were added to whole blood or plasma in varions quantities and a large number of coagulation tests then performed. Considerable differences in the sensitivity of the various assays to the added test substances were evident. It is believed that they were due, at least in part, to the varying dilutions of plasma utilized. The results shed no insight on the mechanisms by which heparin exerts its anticoagulant effects; the inhibitory activity of Polybrene, however, was largely restricted to the stage of thromboplastin generation.Polybrene had a much weaker inhibitory effect than heparin on all tests, and in many assays it was possible to add a standard amount of Polybrene without altering results. The amount chosen was large enough to neutralize any quantity of heparin likely to be present in a sample obtained from a patient during open heart surgery.Factor V could be assayed with accuracy only if heparin was neutralized as the sample was collected. Assay of Factor VIII was successful only if the Polybrene was added after, the step of adsorption with aluminum hydroxide. A one-stage prothrombin assay presented no problems, but assays for Factor VII complex provided distinctly different results from those found in a duplicate sample collected in citrate alone.

Difference in the heparin-neutralizing effect of protamine and polybrene as tested by thrombotest.

Abstract1. Thrombotest as well as other one-stage prothrombin assays is influenced by heparin in vitro and in vivo. After an average dose of heparin intravenously, the effect on thrombotest lasts for about 8 hours.2. When heparin is added to citrated blood in Vitro, polybrene and protamine are both able to correct the prolonged thrombotest time.3. Polybrene is not able to correct the prolonged thrombotest values in blood from heparinized subjects, whereas protamine gives complete correction. Both are able to correct the prolonged thrombin time.4. When 0.06 mg of protamine ml is added to blood from patients receiving combined therapy with heparin and an oral anticoagulant, the effect of heparin is neutralized and thrombotest values reflect only the effect of the oral anticoagulant.

Levels of Blood-coagulation Factors During Anticoagulant Therapy with Phenindione

The complex mechanism of the factors concerned in blood coagulation has been investigated in many ways, not least among these being a study of such factors during anticoagulant therapy, and by the use of plasma from patients treated with anticoagulant drugs in cross mixture experiments with plasma from patients having congenital coagulation defects. Earlier results from such investigations indicated a simple deficiency of prothrombin and VII. It is now known, however, that Christmas factor (Douglas, 1955; Biggs, 1956; Naeye, 1957), plasma thromboplastin antecedent (Naeye, 1957), and Prower-Stuart factor (Telfer, Denson, and Wright, 1956; Hougie, Barrow, and Graham, 1957; Denson, 1958) are all reduced. Factor X* (Duckert, Fliickiger, Matter, and Koller, 1955) is also reduced early in therapy. The existence of the latter factor as a separate entity, however, has not been estab lished, and it is probably an intermediate product of thromboplastin formation. Horder (1958) studied levels of factor VII, Prower Stuart factor, and prothrombin, together with the partial thromboplastin time, and thromboplastin generation in one case receiving anticoagulant therapy with the coumarin derivative phenprocoumon ( marcoumar; 4-hydroxy-3-(1-phenylpropyl)coumarin). Hicks and Bonnin (1959) compared the reduction in plasma thromboplastic factors, the one-stage prothrombin time, and the two-stage prothrombin levels in several cases receiving anticoagulant therapy with different drugs, and concluded that the one-stage assay provided an adequate measure of the reduction in thromboplastic factors.