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3. Laboratory Reagents and Coagulation Assay Procedures*

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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 standardiza- tion 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 moni- toring 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
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