Rosemary Biggs

The Thromboplastin Generation Test

Physiological coagulation is dependent on the formation of a powerful thromboplastin within the blood. It is well recognized that coagulation defects, such as that in haemophilia, are due to a failure of the normal thromboplastin mechanism, but lack of knowledge has hitherto prevented any precise or detailed study of these abnormalities. Biggs, Douglas, and Macfarlane (1953a) have shown that three components, normal plasma treated with AI(OH)3, platelets, and normal serum, react to form thromboplastin. Further analysis has shown that two essential substances, antihaemophilic globulin and factor V, occur in the Al(OH)3-treated plasma, while serum contains two factors, factor VII and the Christmas factor, both of which are required for thromboplastin formation (Biggs, Douglas, and Macfarlane, 1953b). These five components-platelets, antihaemophilic globulin, the Christmas factor, and factors V and VII-react together in the presence of CaCl2 to form a labile thromboplastin as powerful as any so far described. A lack of any one of the five produces a coagulation defect associated with abnormal thromboplastin formation. Factors V and VII are usually thought of as

A Thrombin Generation Test: The Application in Haemophilia and Thrombocytopenia

Much recent research on blood clotting has dealt with prothrombin and factors which influence its activation. In the development of such investigations experimental procedures have become increasingly complex and artificial. They usually involve the use of anticoagulants, often multistage fractionation by precipitation, adsorption or filtration, and, almost invariably, the addition of tissue extracts to promote thrombin generation. These techniques have yielded information of great importance, but they must have a limited application, since they are far removed from the natural process of clotting. Normal blood taken by a clean venepuncture into a glass vessel Clots firmly in a few minutes without any stimulus other than surface contact. It must therefore have some intrinsic source of thromboplastic activity, in the sense that something is formed which converts prothrombin to thrombin. It is clear that a study of this intrinsic thromboplastin is essential to further understanding of normal clotting and its various defects, because the ability of blood to clot without artificial assistance probably determines its haemostatic efficiency. Fractionation techniques can give no information on the earliest stages of natural clotting, which must, initially, be studied in blood which has been altered as little as possible. Estimations of clotting time and of prothrombin consumption can be made on unaltered blood, but because both give only a single figure which is influenced by several different factors, they are of little help in analysing the causes of delayed or deficient coagulation. More information would be provided if a series of determinations of the thrombin content of whole blood were made at intervals during clotting. In 1901 Arthus carried out an experiment of this sort. He took serial samples of dogs blood which was defibrinated by beating, added sodium fluoride, and after centrifuging determined their ability to clot fluoride dog plasma. A simplified technique described in the following paper uses unaltered whole blood from which serial samples are simply transferred to fibrinogen, the clotting time of which indicates the thrombin concentration of the blood at the time of sampling. By plotting thrombin concentration against time, curves are obtained which show the speed of thrombin generation and destruction and thus may indicate the rate of production and the activity of blood thromboplastin. The generation of thrombin in whole blood can only reflect thromboplastic activity if irrelevant variable factors are controlled as much as possible. Variations in prothrombin, antithrombin, and calcium concentration are likely to have a considerable effect on thrombin generation, but they were avoided by the use of normal blood, or blood from cases of haemophilia or thrombocytopenia in which these factors are almost certainly normal.

An investigation of three patients with Christmas disease due to an abnormal type of factor ix

Three patients with Christmas disease whose plasma was shown to have a prolonged one-stage prothrombin time with ox brain thromboplastin have been investigated. These patients have an inhibitor for the reaction between factor X, factor VII, and ox brain extract. The abnormal constituent responsible for this inhibitor appears to be factor IX whuch is functionally inactive but antigenically indistinguishable from normal factor IX. It is proposed that patients might be classified into haemophilia B+ for patients with this defect (Christmas disease+) and haemophilia B− (Christmas disease−) for patients who have classical Christmas disease.

The Errors of Some Haematological Methods as They Are Used in a Routine Laboratory

The quantitative measurements made in a haematological laboratory assess the magnitude of deviations from the normal, and successive readings indicate either the response to treatment or the natural progress of the disease. These figures can be of little value unless the range of chance variation that several laboratory workers may record in one blood sample is less than the difference that the clinician requires to distinguish. It has been usual for the standard of precis on of a method to be set by one expert, whose results, while measuring the minimum error, may bear little relation to ordinary routine practice. In the present study an attempt was made to assess the errors of some common haematological methods as they are used as a routine in laboratories. Those taking part in the experiments were laboratory technicians and doctors of average skill, who carried out the investigations careful:y; any gross technical inaccuraciescan be excluded. It was hoped that the experiments might suggest which techniques were likely to give the most generally reliable results. The methods studied

The Error of the Red Cell Count.

The staffs of haematological laboratories spend much of their time counting red cells. The precision of this estimation is therefore of practical importance, and many experiments have been designed to establish the error of the technique. In spite of this, the most widely divergent views are still held as to the reliability of single observations. In 1881 Lyon and Thoma showed that the standard error of counts made on the same sample of blood was roughly proportional to the square root of the number of cells counted. Thus in a count of 100 cells the standard error would be roughly ./ 100, and repeat counts on the same sample might by chance vary from 80 to 120 cells, or, in counts of 500 cells, the standard error would be approximately V/ 500 or 4.5 per cent. In 1906-7

The Reaction of Haemophilic Plasma to Thromboplastin

Two well-known observations on the clotting defect of haemophilic blood would seem to be of basic importance. The first is that haemophilic blood responds quite normally to a strong suspension of tissue thromboplastin; the second, that it will clot in practically the normal time on the addition of a small proportion of normal plasma. The normal response to thromboplastin naturally suggests that the part of the clotting mechanism which is set in motion by thromboplastin is in normal working order. Certainly no defect has been demonstrated in haemophilic prothrombin, fibrinogen, or calcium, and the various factors recently described as being concerned in prothrombin conversion, such as Factor V (Owren, 1947) and serum prothrombin conversion accelerator (Alexander and de Vries, 1949), are stated by these respective authors to be normal in haemophilia. The essential defect in haemophilic blood, therefore, seems to be its inability to clot normally in the absence of added thromboplastin, a defect which is largely corrected by the addition of a small amount of normal plasma. Since normal blood clots rapidly in glass, even when obtained with a minimum of contamination with tissue fluid, it must be supposed that there is some intrinsic mechanism for the production of thromboplastic activity. This matter has been discussed by many workers, and two current views are put forward by Brinkhous (1947) and Quick (1947) respectively. These authors agree in supposing that intrinsic thromboplastic activity develops on contact with a foreign surface, and depends on interaction between the platelets and a plasma factor. In Quicks view it is the plasma factor, thromboplastinogen, that is the inactive precursor of thromboplastin and which is activated by a platelet factor. Brinkhous considers that the platelets themselves are the source of thromboplastin, and that they are activated by being broken up by the plasma factor, which is a thrombocytolysin. It is agreed by both that the platelets are normal in haemophilia, and that it is the plasma factor which is deficient in this condition. Normal plasma contains enough of this antihaemophilic factor to be able to correct the deficiency. Ignoring the slight divergence of views as to the mode of action of the anti-haemophilic factor, it seems to be accepted that its deficiency in haemophilia results in a deficient production of intrinsic thromboplastin, such thromboplastin presumably having a similar action to that of the more familiar tissue thromboplastin. The normal response …

Estimation of Prothrombin in Dicoumarin Therapy

The concept of prothrombin as the specific precursor of thrombin dates from the so-called classical theory of blood coagulation put forward by Schmidt (1895) and Morawitz (1905). This theory, which considers that four factors, thromboplastin, prothrombin, calcium, and fibrinogen, are concerned in the formation of fibrin, has been the basis of most modern work. The theory was of more academic than practical importance until the discovery of vitamin K and the use of dicoumarin in treatment of thrombosis, both of which required the quantitative estimation of prothrombin. The available methods are all based on the assumption that the classical theory is fundamentally sound. Of the methods of prothrombin estimation that have been developed, none can measure prothrombin directly and therefore infer its concentration from observations on thrombin. The one-stage technique of Quick (1942) has been the most commonly used because it is simple, easily performed, requires little in the way of special reagents, and gives results which are in general agreement with clinical experience. The method consists of adding an optimum amount of brain thromboplastin and calcium to oxalated plasma, under which conditions the clotting time of the mixture is assumed to be proportional to its prothrombin concentration. The basis of this supposed relationship is the literal acceptance of the classical theory. If three of the four variables are controlled, then variations in the fourth must determine any alteration in the rate of fibrin production, which is in turn indicated by a change in the clotting time of the mixture. In practice thromboplastin and calcium are controlled, if possible at or near their optimum concentrations, and fibrinogen variations found in the ordinary clinical material are usually not of sufficient magnitude to influence the result. The remaining variable is therefore prothrombin. The method is essentially dynamic in principle, depending entirely on the rate of thrombin generation and not on the amount of thrombin that may finally be produced. It depends also on the assumption that there are no uncontrolled factors other than prothrombin that might modify this rate of thrombin generation. It is now known, of course, that this is not the case. Not only may the reaction time of fibrinogen vary, but accelerators and depressors of thrombin generation are present in normal plasma, and variation in these accelerators and depressors in pathological conditions may alter the results of this test. Quick (1942) himself has been the first to recognize this. The practical implications of the fallacious nature of this method are difficult to assess at present, but no alternative to it is available for routine use. Though theoretically the two-stage method appears to be on much firmer ground, since it seeks to estimate the total amount of thrombin generated, it is too complex for ordinary routine use. There are, moreover, many indications that its results may be equally fallacious. In dicoumarin therapy one component of prothrombin (prothrombin B of Quick, 1947) is deliberately lowered, and the responsibility for a reduction to haemorrhagic levels rests largely with the pathologist. A reliable method of prothrombin determination is therefore essential. There has recently been much discussion as to which of the modifications of the one-stage technique give the best results (Marsh, 1947, 1948; James, 1948; Cleland, 1947; Pivawar, 1947; Lempert, 1948; Canti and Robertson, 1948).

Laboratory tests for incipient thrombosis.

The use of anticoagulant therapy in the treatment of thrombosis is now widespread, and there is little doubt of its value. It is therefore not surprising that many laboratory workers have attempted to find tests to demonstrate hypercoagulability of the blood, so that, in susceptible cases, anticoagulants might be used to prevent the onset of thrombosis. It appears that blood does not clot solid in the vessels of normal people because the surface with which it is in contact does not readily liberate the thromboplastin which is essential for the conversion of prothrombin to thrombin. When blood is withdrawn from the body and maintained at 370 C. in a glass tube, clotting occurs in five to 10 minutes because contact of blood with the glass surface leads to the liberation of a powerful thromboplastin. After coagulation of normal blood in a glass tube most of the prothrombin activity disappears from the serum; the extent to which this hasoccurred may be an index of the efficiency of the blood thromboplastin system. When blood is held at 370 C. in tubes coated with silicone, it takes upwards of 20 minutes to clot and only a fraction of the prothrombin activity disappears: it is thought that this is because the silicone surface does not readily promote the liberation of thromboplastin. Thus it is probably true to say that clotting of normal blood is limited by the extent to which free, active thromboplastin can be formed. The diagnosis of a prethrombotic state may therefore depend on demonstrating some abnormality of the blood thromboplastin system. Since there were at the time when these investigations were made no specific tests for the investigation of this system, indirect methods were used. Three techniques are available. The first method is to compare the clotting time of whole unaltered