3. Laboratory Reagents and Coagulation Assay Procedures*

STANDARDIZATION OF DIAGNOSTIC MATERIALS

3. Laboratory reagents and coagulation assay procedures*

E. A. LOELIGER 1

This paper reviews current methods ofstandardizing laboratory investigations in blood clotting, points out their shortcomings, and suggests ways of improving them. Procedures requiring standardization include the diagnosis of haemorrhagic diathesis and thrombotic tendency; the quantification of liver function; and monitoring of the effect ofhaemostatic replacement therapy and therapeutic anticoagulation. Care in collecting, storing, and handling bloodfor investigation is ofprimary importance. Achievements in the standardization of reagents are reviewed and examples of standardized methods are given. Although knowledge of the haemostatic process and experimental methods for its investigation have reached an advanced stage, their clinical application is still unsatisfactory owing to the inadequacy of national and international regulations. To improve the situation, one or more internationally approved laboratories for standardization and quality control, operat- ing according to rules elaborated by experts in the field, should be designated in each country. The needfor international reference laboratories is urgent.

FIELD AND LEVEL OF INVESTIGATION Thrombotic tendency Haemorrhagic diathesis Conclusive data are not available to substantiate by means of laboratory tests. A In the first instance, the mechanisms of primary such a tendency screened. The simple increase in the number of platelets is known haemostasis and blood clotting are to be accompanied by an increased number of former is accomplished by determining the (primary) thromboembolic events. It has been asserted that a bleeding time (Ivy et al., 1941; Mielke et al., 1969), positive ethanol gelation test-an easily performed counting the platelets (Ludin, 1952), and inspecting a good indicator of immi- the blood film. The latter involves measuring the screening procedure-is time, and nent thrombosis. A strongly increased Factor VIII partial thromboplastin time, prothrombin level or decreased fibrinolytic activity also contri- fibrinogen level, as well as testing for fibrin(ogen)- of the clot butes to the formation of thrombi. Studies in hetero- degradation products and urea solubility zygotes for the antithrombin Ill deficiency have (clot retraction is of lesser importance). that an insignificant If the results of screening together with the case shown convincingly apparently in- shortage of this naturally occurring circulating anti- history yield insufficient information, laboratory coagulant is associated with recurrent thrombo- vestigation is extended by an assessment of separate determinations also call for platelet functions and coagulation factors (Biggs, embolic episodes. These 1972; WHO Scientific Group on Inherited Blood specialized laboratories. Clotting Disorders, 1972). These procedures require Other procedures specialized laboratories. Quantification of the liver function and the monitoring of anticoagulant therapy may be performed * Presented at the International Conference on Stan- dardization of Diagnostic Materials, Center for Disease at the routine level by means of the prothrombin Control, Atlanta, Ga., USA, 5-8 June 1973. time test. However, for the laboratory assessment of ' Head, Laboratory Thrombosis Research, Haematology the patient's response to replacement therapy, Section, Department of Medicine, University Hospital, Leiden, Netherlands. special laboratory facilities are mandatory.

3073 -727 728 STANDARDIZATION

UNDESIRABLE EFFECTS OF INSUFFICIENT Gurd, 1972). For storing blood or plasma at room STANDARDIZATION temperature for several days, the addition of small amounts of sodium azide (0.1 mg/ml plasma) has Collection of venous blood been shown to prevent bacterial growth (E. A. L., Laboratory investigation of blood clotting begins unpublished observations, 1969). with the collection of venous blood. Prolonged venous pressure results in a 10-30% increase in the Dilution protein-bound enzyme activity and in platelet num- The dilution step in the assay procedure is of bers. In blood that is slowly drawn or (as in the case particular importance, because it introduces so many of capillary blood) not easily obtained, the clotting unexpected phenomena-e.g., at low ionic strength, mechanism may be activated. This results in exces- the fibrin clot, under the influence of thrombin, sively high coagulation factor activity and in a forms more rapidly and more easily than when the falsely low number and disturbed function of plate- ionic strength of the reaction medium is " normal ". lets. Therefore, blood for the investigation of blood A second example: the magnitude ofthe final dilution clotting should be taken only by highly experienced of the plasma to be investigated has proved to be of personnel. special importance in the control of oral anticoagu- Decalcification ofblood lation, under which conditions a competitive inhi- bitor-the so-called protein induced by vitamin K Sodium citrate should be used at a strength of absence and antagonists (PIVKA)-enters the cir- 110 mm. Salts of oxalic acid display a stronger culation. The final concentration is of great impor- avidity for calcium ions, resulting in a rapid loss tance for the result obtained with PIVKA-sensitive of Factors V and VIII. The use of EDTA-an even methods, such as the thrombotest and tests using stronger calcium binder-gives rise to a rapid loss human-brain thromboplastin. When PIVKA-sensi- not only of Factor V and Factor VIII activity but tive procedures are used to test blood rather than also of the clottability of fibrinogen by thrombin, in plasma, the correction factor for the venous haema- addition to the complete disaggregation of blood tocrit is much smaller than might be expected platelets. from the differences in the amount of plasma cal- Stabilization of the labile factor culated for various haematocrits. Another example of the importance of the dilution step is the strength For the stabilization of the so-called labile factor of the in the (Factor buffered solution is needed. phospholipid suspension used partial V) decalcifying time as HEPES buffer has to be thromboplastin test, high-activity suspensions proved particularly useful easily suppress the activity of the anti- (Zucker et al., 1970c). circulating coagulant occurring in autoimmune diseases. As a Containers andpipettes consequence of the use of phospholipid suspensions Containers and pipettes in which blood and plasma of different strengths for the assessment of individual are stored, transported, diluted, and otherwise hand- coagulation factors, acquired isolated coagulation factor may be led, must be contact-free (siliconized or made of deficiencies erroneously diagnosed plastic) and clean. Only for the final step of pipetting, (Largo et al., 1972; Castro et al., 1972). Finally, in performed immediately before the recording of the the procedure for thromboplastin calibration (Biggs & Denson, 1967b), the final dilution of the reaction, is it possible to use disposable (micro) glass thrombo- pipettes. Yet, despite this precaution, slow, although plastin to be calibrated must be exactly defined, slight, activation of coagulation factors is regularly because the sensitivity of a thromboplastin rises as observed during the first 6-12 hours after blood the dilution is increased. sampling. Centrifugation Storage temperature The centrifugation step is also highly important, The blood to be investigated should be stored at not only in platelet function tests, but also in coagu- 18-23°C. At low temperatures (0-40C) the activity lation research-e.g., after adsorption of the factors of Factor VII may increase to many times its normal of the prothrombin complex where high-speed centri- level through cold-activation of plasma kallikrein fugation (30 min at 20 000 g) is needed to eliminate (Gj0nnaess, 1972). The addition of kallikrein inhi- the residual aluminium hydroxide (Loeliger et al., bitors does not suppress this activation (Brozovic & 1973). COAGULATION PROCEDURES & REAGENTS 729

Reagents (Zucker et al., 1970a). The choice of the most The example of the large differences in the pro- appropriate apparatus from among the large variety longation ratio (patient's coagulation time: normal of automatic clot timers is difficult. The fibrometer coagulation time) obtained with the various thrombo- system has become the most frequently used instru- plastin preparations in use-differences that are far ment in the USA. Photoelectric clot timers have also from eliminated by translating the prolongation been found useful (Sibley & Singer, 1972). Schnitt- ratio into a conventional percentage of activity ger's coagulometer is particularly suitable for the (Table 1)-is sufficient to demonstrate the urgent assessment of Factors VIII, IX, XI, and XII (Velt- need for standardization in this respect. kamp et al., 1968). Reading ofresults Observer bias With regard to the laboratory record of the results The type of reading used for the registration of obtained-i.e., the observer's bias-the observer clotting times has been shown to be important. should know that results for haemostatic parameters Particularly where the clotting process is relatively are subject to wide biological variations and experi- slow-e.g., in the method for assessing the so-called mental error. Biological as well as analytical coeffi- prothrombin and proconvertin complex (P & P cients of variation may amount to 25 % (Veltkamp method according to Owren & Aas, 1951)-well- et al., 1968; Goldenfarb, 1971). Thus unexpected standardized automatic reading greatly improves the results are often obtained, and these must be recorded comparability of results obtained in different labora- and taken into account. tories (E. A. L., unpublished observations, 1972). Mechanized coagulometry for Quick's original one- Other factors stage test, in skilled hands, does not perform signi- Certain other factors, such as the temperature at ficantly better than manual testing (Fewell et al., which the reaction takes place and the successive 1972); automatic reading is, however, more econo- steps of the test procedure, should be defined exactly, mical. Although coagulometers may measure shorter the latter particularly in regard to the amounts of coagulation times than hand reading, prothrombin reactants used, the order of addition, and the time time ratios (specimen test in s: control test in s) of incubation of the different reactants. If the result appear to be independent of the type of reading of an assay is expressed in relation to a normal reference sample, the two should be investigated in Table 1. Prolongation ratios a obtained with various parallel because of instabilities of the biological assay procedures, and their equivalents in conventional material. percentages (according to Biggs & Denson, 1967a) STANDARDIZATION OF REAGENTS AND METHODS Assay procedure Prolongation ratio percentage Relatively poor results have been obtained, even at the expert level, in the standardization of methods Quick's procedure and reagents, although it is known that inadequate Simplastin b 1.45-2.1 18-30 standardization leads to after-effects that are extreme- human brain (BCT) 2.5 -4.0 15-26 ly dangerous for the individual patient. There can P & P method be no doubt that the aimost total lack of formal & 2.8 regulations pertaining to standardization and quality (Owren Aas, 1951) -5.0 control of laboratory investigations is responsible Ware & Stragnell, 1952 1.6 -2.4 10-20 for the present situation. Only reagents that are used Simplastin Ab' 2.5 -4.6 as drugs, e.g., heparin, thrombin, and thrombolytic Thrombotest C 2.7 -4.1 5-10 agents, are subject to legal controls (pharmacopoeias). Most of the available coagulation assay procedures International percentage 7-14 are bioassays. Thus the active material to be assessed can be estimated only by observing the response a Prolongation ratio = patient's coagulation time: normal that it evokes in vivo or in vitro and by comparing coagulation time. the a amount b Warner Lambert; Nyersey; etc. it with response caused by known c Nyegaard. of the active material and dilutions of it. The known 730 STANDARDIZATION amount must therefore refer to well-defined standard recent international collaborative study, sponsored reference material.1 by WHO, did not disclose major discrepancies, in To illustrate current methods of standardizing vitro, between heparin from different sources (Bang- clotting reagents, the examples of heparin, blood ham & Woodward, 1970). Examples of how to clotting Factors VIII and IX, and tissue thrombo- compare heparin from different origins in vivo are plastins are given below. Other tests and reagents to be found in the recent literature (Gomez-Perez, will be referred to only briefly. 1972; Baltes et al., 1973). Fortunately, no significant differences between hog intestinal mucosa and bovine Heparin lung heparin has been found. Nevertheless the current Heparin is issued with a potency value in units International (intestinal mucosa) Standard has re- per weight, the formula 100 IU/mg being generally placed the 1958 bovine lung standard, since about applied, although the activity per mg of recent 80 % of all commercially available heparin prepara- preparations is as high as 160 IU. tions are now made from pig intestine (Bangham & Standardization ofthe biological activity ofheparin Woodward, 1970). is based solely on its coagulation-inhibiting effect in vitro. Points of references are: Blood coagulation Factors VIII and IX Internationally: a standard established and ap- For patients suffering from haemophilia A and B proved by WHO (the latest in 1973); blood products of fairly constant quality are being Nationally: the British Pharmacopoeia and the obtained, provided that standard procedures of pre- United States Pharmacopeia standard preparations; paration are followed strictly. The in vivo activity of Locally: working standards or the so-called house cryoprecipitate concentrated Factor VIII can easily standards used by commercial firms to calibrate the be predicted from the activity assessed in vitro batches that they produce. (Meyer et al., 1967) except that, for Factor IX Heparin appears to have been satisfactorily concentrates, it is dangerous to rely on such an standardized, judging by its 30 years of widespread assessment. Many Factor IX concentrates contain use without reports of major difficulties by either activated Factor IX, which disappears rapidly in clinicians or pathologists. Yet, considering not only vivo (Bruning & Loeliger, 1971). Substitution therapy that heparin is prepared from different tissues (e.g., for major surgery, particularly in haemophilia B bovine lung; intestinal mucosa of various animals, patients, must be checked once or twice daily to notably the pig) and consists of mixtures of compo- ensure haemostatic levels of Factor IX (Biggs, 1972). nents with many divergent chemical and physical Diagnosis, the control of treatment, and genetic properties, but also that its calibration is based on an counselling for possible carriership of the haemo- assay procedure devised some 20 years ago, the philic gene (Veltkamp et al., 1968) require both existence of rather large differences in biological highly standardized assay methods and reference activity between different batches would hardly be materials. For the assessment of Factor VIII, natio- surprising. The repeated warnings given during the nal reference centres can rely on an international past 10 years (for references see Jaques, 1972), reference preparation that has been available since and observations of marked differences in clinical 1970 at the WHO International Laboratory for Bio- effectiveness between two preparations (Goldberg logical Standards (Bangham et al., 1971). This re- et al., 1972), should not be underestimated. Other ference preparation, containing purified Factor VIII authors have reported that subjects treated with derived from cryoprecipitate, has been shown to heparin prepared from porcine gut require 25 % less fulfil the requirements of a long-term standard. protamin sulfate as an antidote than those treated It is important to keep in mind that freeze-dried with lung heparin (Sekhar et al., 1971). These obser- plasma preparations are less stable, but-like any vations call for a reassessment of the standardization other properly prepared and stored plasma prepara- procedure (Jaques & Kavanagh, 1972), although a tion-can serve as a working standard for at least one year. The reason why national standardization 1lInternational standards approved by WHO are made is developing so slowly may be that most countries available by the WHO International Laboratory for Bio- logical Standards, National Institute for Biological Standards do not have national expert panels and reference and Control, Holly Hill, Hampstead, London, England. laboratories and individual laboratories can rely on Working standards are produced and made available by house standards- national institutes; and house standards refer to preparations pooled normal plasma-so-called produced locally. stored at -70°C (Aronson, 1972). COAGULATION PROCEDURES & REAGENTS 731

For haemophilia B-i.e., treatment with Factor IX been developed with which the " therapeutic range " -a subcommittee of the International Committee is defined (Miale & Kent, 1972). The Center for on Haemostasis and Thrombosis (ICHT), still active- Disease Control in Atlanta (CDC) has also contri- ly exploring the feasibility of in vitro assessment of buted to the standardization of the one-stage pro- Factor IX (concentrates), is attempting to define thrombin time procedure: it established that com- one or more international reference preparations. mercial rabbit tissue reagents are sufficiently sensitive The first results were expected to be available by the to provide safe anticoagulant control, although they end of 1973. It may already be concluded that future are less sensitive than the human-brain thrombo- reference material should be derived from plasma and plastin and thrombotest reagent in assessing the anti- should not contain activated Factor IX. Hence, a coagulant effect (Zucker et al., 1970b). test for activated Factor IX must also be developed In Great Britain, Poller has been responsible for to exclude possible contaminations with activated successful national standardization based on the so- Factor IX in the sample to be tested. A nonactivated called British comparative thromboplastin (BCT) and partial thromboplastin time might serve this purpose referred to as the British system (Poller, 1964). (Aronson, 1972). This system also implies a definition of the " thera- From the results of the collaborative studies on peutic range " (Poller & Loeliger, 1969; Bailey et al., standardization of the Factor VIII and IX procedure 1971). published so far, it appears that reliable methods On the international level, the so-called equivalent do exist: a highly standardized (simplified) two- ratio method (Biggs & Denson, 1967b) has been stage assay procedure (Denson, 1967) and an equally developed by ICHT. The validity in practice of well standardized one-stage assay procedure (ori- the assumptions made by Biggs and Denson has ginally introduced by Hardisty & Macpherson, 1962, been proved (Loeliger & Hemker, 1969). Five ICHT- and modified by Veltkamp et al., 1968). approved trial reference thromboplastins are maintained at the Division of Biological Standards of the Tissue thromboplastins British National Institute of Medical Research (Biggs Thromboplastin calibration is one of the main & Bangham, 1971) for the calibration of national concerns of standardization of the prothrombin and commercial thromboplastin preparations. time test procedure, since it permits conversion to an The currently recommended calibration procedure international scale of the highly divergent results (Denson, 1971) requires freshly prepared plasma obtained with the various laboratory methods and from 4 normal individuals and from at least 20 thromboplastins in use at present for the control patients stabilized on oral anticoagulants; a cali- of coumarin-induced hypocoagulability. bration table is used for the conversion of the results The ultimate goals of standardization are to into international terms. Since this procedure is achieve an internationally accepted definition of complex, ways of simplifying it are being sought. the so-called therapeutic range of hypocoagulability Lyophilized pooled plasma substituted for fresh in patients on oral anticoagulation therapy (Loeliger, plasma gave promising results (Bangham et al., 1973). 1972) and to determine how this is expressed in the A given set of such plasma has been shown to re- results of both a standard thromboplastin and a main useful even after heating at 37°C for several standard test (and also, if required, in the results months (E. A. L., unpublished observations, 1972), obtained by a given thromboplastin in a particular and artificially prepared lyophilized plasma may also test employed locally-although this might be diffi- be useful, at least for the calibration of PIVKA- cult to achieve, since certain thromboplastins and/or insensitive thromboplastins. Lyophilized plasma is test procedures might prove to be unsuitable for insufficiently stable, however, to be used as long-term anticoagulant control, e.g., because of the marked reference material (Brozovic et al., 1971a, 1971b), insensitivity to the defect produced by anticoagulants although there is good reason to believe that it can in the blood of the treated patients). serve as short-term reference material (Miale & Kent, In the USA, the College of American Pathologists 1972). adopted the principle that standardization should be With the development of standardization by means based on the performance, in a strictly defined pro- of thromboplastin and plasma reference material, the thrombin time assay procedure, of reagents in re- ultimate goal of standardization may be at hand, lation to clearly defined reference materials (Miale as many centres with long experience already appear & LaFond, 1969). Plasmatic reference material has to be recommending therapeutic ranges closely simi-

7 732 STANDARDIZATION

Table 2. Therapeutic ranges recommended by various For the diagnosis of overt diffuse intravascular research centres (data according to Biggs & Denson, 1 967a) clotting and fibrinolysis, immunological test procedures are usually applied. The qualitative and Therapeutic range quantitative immunodiffusion and precipitation tests are Centre Biggs/Denson proposed widely used (Ouchterlony, 1964; Mancini et al., ratioratio ~~internationalpercentage 1965). More sensitive quantitative tests are a well- standardized tanned red cell haemagglutination in- hibition immunoassay (TRCHII) (Merskey et al., Basle (human brain) 1.85-2.6 7-14 1969 and 1971) and, to a lesser degree, the staphylo- Leiden (thrombotest) 2.6 -4.0 7-13 coccal clumping test (SCT) (Hawiger et al., 1970), London (rabbit brain) 1.6 -2.0 9-14 the results of which are expressed in fibrinogen equivalents. For the differentiation of intravascular Manchester (human brain) 1.86-3.0 8-17 coagulation and fibrinolysis, which might have impoi- New York (rabbit tant therapeutic repercussions, turn-over studies and brain) 1.5 -2.0 7-13 immunochemical tests of fibrin(ogen)-degradation products are under study (G. Izak, unpublished observations, 1973). Standardization of the SCT has been proposed lar to those shown in Table 2 (Biggs & Denson, (Niewiarowski & Thomas, 1971). The results ob- 1967a). The optimum range appears to be 7-14% tained with these techniques in different laboratories (international scale), and this has given excellent compare rather well, even when the same reagents clinical results, also in atherothrombosis (Loeliger are not used (Sherry & Johnson, 1971). The Sub- et al., 1967; Hamming et al., 1965; Meuwissen et committee on Fibrinolysis and Thrombolysis of al., 1969). ICHT has also considered standardization of fibrin- As there is insufficient information on the relative (ogen)-degradation products (Sherry & Johnson, merits of the various types of plasma and thrombo- 1971). plastin reference materials, an international Colla- In general, increased or decreased fibrinolytic borative Study on Prothrombin Time Standardiza- activity can be tested by the euglobulin lysis test. tion was planned for 1973-74 under the auspices However, this test is of limited value as the result of the International Committee on Standardization depends on so many imponderables; furthermore, the in Haematology (ICSH), with the CDC, Atlanta, lytic activity of highly diluted plasma cannot be as the organizing centre. representative of undiluted blood. In clinical practice It is worth mentioning that the calibration of the test should be abandoned. If the activity of the thromboplastins differs from the classical biological naturally occurring activator is to be assessed in standardization only in that plasma reference mate- experimental work, more specific tests should be rial prepared from patients is used, the response of used, including the assessment of fibrinolysis-in- reference thromboplastin to this material being hibiting globulins. taken as the baseline. The definition of plasma The standardization of fibrinolytic agents and reference material in terms of coagulation factor assay methods was assigned mainly to a working activity has been suggested (Loeliger et al., 1970). subcommittee of the Committee on Thrombolytic Agents (CTA) of the US National Heart Institute. Other tests and reagents Streptokinase was standardized first (Bangham & Testsfor intravascular coagulation, fibrinolysis, and Walton, 1965), and in the early '60s a lyophilized thrombolysis. One of the first laboratory signs of research reference preparation (working standard) diffuse intravascular coagulation, particularly in the of urokinase, the potency of which was defined by case of a rapidly growing thrombus, may be soluble CTA, was developed and was subsequently approved fibrin detected by the protamine sulfate and ethanol by WHO. Unfortunately, a collaborative study car- gelation test (Godal & Abildgaard, 1966; Lipinski & ried out in 1967 under the auspices of WHO has not Worowski, 1968; Konttinen et al., 1972). The Biogel yielded sufficient data to establish the material in column method (Fletcher & Alkjaersig, 1971) seems question as an international urokinase standard promising but is not yet suitable for routine use. (WHO Expert Committee on Biological Standardi- Imperfect venepuncture leads to positive results. zation, 1969). COAGULATION PROCEDURES & REAGENTS 733

The National Heart Institute also prepared stan- tested) have been developed, and these are especially dard batches of such substrates as plasminogen, convenient in clinical emergencies (Clauss, 1957; fibrinogen, a-casein, and the methyl ester of acetyl- Vermeylen et al., 1963). Although they are highly lysine, which can be obtained on request and used useful in practice, biological tests cannot be con- in working-standard methods for the assay of plas- sidered as specific either: falsely low values will be min, plasminogen, and urokinase (Johnson et al., obtained in case of circulating fibrin(ogen)-degrada- 1969). Glycerol-activated plasmin has been under tion products. study since 1971. Human plasminogen is assayed Thrombin activity is currently expressed in NIH by activating it to plasmin with 200 units of strepto- units. Research reference preparation 66/305, con- kinase or 2 000 CTA units of urokinase. The amount taining 200 units per ampoule, is available from the of plasmin produced is determined by comparison WHO International Laboratory for Biological Stan- with a standard plasmin curve. The potency of dards. A collaborative study under the auspices of plasmin(ogen) has been defined in CTA terms. WHO was started in 1971 to develop an international standard preparation (Sherry & Johnson, 1971). This Bleeding time, platelet adhesiveness, and platelet preparation is now ready for approval (A. J. John- aggregation. An international cooperative study on son, unpublished observations, 1973). the bleeding time test, designed to set standard The assessment of antithrombin III activity is of conditions (Born & Mason, 1971), has not been great importance for the diagnosis or exclusion of carried out yet. The need for such a study has even a thrombotic tendency. The acquisition of a well- been doubted, because of the availability of well- standardized and reproducible assay method is defined procedures such as the tests according to therefore most welcome (Abildgaard et al., 1970). Ivy (Ivy et al., 1941) and Borchgrevink (Borchgre- Standardization of the test and its comparison with vink, 1958), or one of their modifications (Mielke immunological assay procedures are being investi- et al., 1969). gated at the WHO International Laboratory for The results obtained from a cooperative study on Biological Standards. platelet adhesiveness were rather disappointing: the result of the Salzman test for platelet adhesiveness Determination ofseparate coagulationfactors. One- to glass beads does not make it substantially easier stage assay procedures appear to be highly suitable to diagnose von Willebrand's disease (Murphy & for standardizing the determination of the separate Salzmann, 1972). coagulation factors. So far no attempts have been The standardization of the turbidimetric method made to standardize any of these tests. The problem, for quantification of platelet aggregation is still in however, is not too difficult to solve once the princi- the pilot-study stage. Optimum test performance ples of thromboplastin and partial thromboplastin requires the use of freshly prepared platelet-rich standardization have been elucidated. More elabo- plasma. There seems to be little difference between rate techniques will be needed to assess activated intraindividual and interindividual variation, both coagulation factors (particularly Factors VII and of which are large (Goldenfarb et al., 1970). It has IX). Finally, a standardized test for PIVKA has to been established that a correction should be made be developed, for which purified staphylocoagulase for the anticoagulant when haematocrit values are should be available. abnormal. Plasma should be prepared by centrifu- Systems ofstandardization gation at 1 000 rev/min for 15 min, kept at room temperature, and used within one hour (Born & Local. It will be necessary to adopt methods of Zucker, 1972). investigation referred to in this paper and recommended by experienced authors (Biggs, 1972) until Fibrinogen, thrombin, and antithrombin III. Stan- approved standardized methods become available dardization of the quantification of clottable fibrino- at the local level. For reagents, calibrated working gen (Factor I) constitutes a problem in clinical che- or home standards should be applied. In addition, mistry when the amount of washed fibrin threads is a stringent quality-control system (Whitby et al,. assessed gravimetrically or chemically. It should be 1967) is indispensable. realized, however, that contamination of the washed fibrin threads with other proteins leads to inconsis- National. Regulations at this level are scarce. In tent results (Beck, 1970). Biological assay procedures the USA, NIH provides a series of working standards (thrombin clotting times of the diluted plasma to be and prescriptions for working-standard methods, and 734 STANDARDIZATION prothrombin time proficiency testing programmes that has been accumulated in the fields of haemo- are operated. stasiologic methodology and coagulation biochemis- The British system of anticoagulant control try. Many laboratories are unaware that the tests (Poller, 1964), maintained under the auspices of and techniques they use are obsolete and unreliable. the British Anticoagulant Panel of the British Com- The reason for this situation is an almost total lack mittee for Standardization in Haematology, conti- of national regulations. nues to improve oral anticoagulation on the natio- The situation could be rapidly improved if national level, and it is hoped that the system will be nal public health inspection services, upon the re- introduced into other countries (Bailey et al., 1971). commendations of expert committees of national Dr Poller's centre 1 has recently been approved by haematology societies and/or colleges of pathologists the British Anticoagulant Panel as a national with industrial representation, were backed by legis- reference laboratory. lation for the use of standard reference material and The Dutch system of standardization, which has methods. For the coordination of these activities, been introduced under the auspices of the Dutch Fed- private or governmental national reference labora- eration of Thrombosis Services, relies on thrombo- tories would need to be designated. These would be plastin calibration with lyophilized plasma pre- responsible for providing all the facilities for the parations from healthy individuals and patients standardization of methods and reagents for the indi- (Loeliger et al., 1972). Artificially prepared normal vidual laboratories, and would operate according to and abnormal blood and plasma will be used for internationally approved directives. They would have quality control. to introduce monitoring systems and proficiency- International. No recommendations concerning testing programmes to safeguard the quality of stan- standard methods have ever been made at the inter- dard preparations and to improve the performance national level, with the exception of working stan- of diagnostic tests. The regular licensing of indivi- dard-methods to be used for the assessment of dual laboratories should be considered. fibrinolytic agents (Johnson et al., 1969). With International facilities for control would need regard to reagents, a small series of well-standardized to be created as a consequence of national regula- reference materials is available at the WHO Inter- tions: international reference laboratories should be national Laboratory for Biological Standards. There designated upon the recommendations of expert is good hope that, for the one-stage prothrombin panels instituted by existing international groups. time test, internationally approved standard material These international laboratories would calibrate and and test prescriptions will be available in due monitor national reference material and commercial course through the cooperative activity of ICSH preparations, and organize international proficiency and ICHT. testing trials to define the status of the field at the international level. Finally, an international clearing CONCLUSIONS house could coordinate the standardization activities and promulgate the results achieved. Standardization of methods for the investigation of the clotting process and of the reagents used has 1 National Anticoagulant Control Reagents Laboratory, lagged far behind the rapidly increasing knowledge Withington Hospital, West Didsbury, Manchester, England.

ACKNOWLEDGEMENTS

The author expresses his sincere thanks to Professor G. I. C. Ingram, Department of Haematology, Louis Jenner Laboratories, St Thomas's Hospital and Medical School, London, and to many other participants in the WHO/CDC meeting on Standardization of Diagnostic Materials, held in Atlanta, Ga., USA, 5-8 June 1973, for their constructive comments and suggestions. COAGULATION PROCEDURES & REAGENTS 735

R,]SUMt STANDARDISATION DES REACTIFS ET DES TECHNIQUES DE LABORATOIRE DANS LE DOMAINE DE LA COAGULATION SANGUINE

La standardisation des techniques de laboratoire dans le facteur VIII, utilis6s non seulement au laboratoire, le domaine de la coagulation sanguine concerne d'une mais aussi pour le traitement de certaines affections, ont part le diagnostic des diatheses hemorragiques et de la d6ja fait l'objet d'une standardisation a l'echelle inter- propension aux thromboses et d'autre part lWtude de la nationale. Pour d'autres, les etudes a ce sujet sont fonction h6patique, la surveillance des traitements anti- actuellement en cours. C'est le cas notamment pour le coagulants et le controle de la r6ponse des malades aux facteur IX, la thromboplastine tissulaire, la profibro- therapeutiques substitutives dans les troubles de la coa- lysine, les produits de d6gradation de la fibrine et l'anti- gulation. thrombine III. La standardisation des methodes apparait necessaire L'auteur expose l'etat actuel de la question et plaide aux divers stades des investigations: a) prelevement du en faveur d'une r6vision r6guliere des processus de sang veineux, materiel i utiliser pour sa conservation et standardisation allant de pair avec les progres de nos son transport au laboratoire; b) manipulation et traite- connaissances concernant les ph6nom6nes de coagula- ment de l'echantillon au laboratoire meme: risques d'action sanguine et le perfectionnement des techniques. tivation, ajustement de l'equilibre ionique et du pH, II met I'accent sur l'importance de la mise en application dilution, centrifugation, temp6ratures de conservation et de systemes de standardisation aux 6chelons international, de r6action, execution des divers temps de la r6action; national et local. Un contr6le international et une regle- c) lecture et interpr6tation des resultats. mentation nationale sont indispensables pour obtenir et Quelques resultats ont e obtenus en ce qui regarde maintenir un niveau de qualite satisfaisant des diagnostics la standardisation des reactifs. Un petit nombre d'entre de laboratoire dans la pratique courante. eux, comme l'heparine, la streptokinase, l'urokinase et

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