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C H A P T E R 129

LABORATORY EVALUATION OF HEMOSTATIC AND

THROMBOTIC DISORDERS

Menaka Pai

This chapter provides a practical approach to the laboratory evalua- of zymogens that become serine proteases. This system functions in
tion of hemostatic and thrombotic disorders. Any assessment of concert with platelet activation and fibrinolysis (Fig. 129.2). However,
hemostatic or thrombotic disorders must start with a thorough clinical laboratory testing of coagulation proteins is not based on this
history and physical exam. These can provide clues to guide subse- current understanding—it follows the original Ratnoff-Davie-Mac-
1,2
quent laboratory testing, diagnosis and management. farlane surface-activated coagulation cascade hypothesis. These tests
Physiologic hemostasis is a complex interplay of cellular or plasma attempt to mimic in vivo processes by carrying them out in vitro,
elements: the adhesion of platelets to damaged endothelium, the and thus do not capture the true complexity of physiologic hemosta-
aggregation of platelets to form a temporary plug, the successive sis. They are still useful in diagnosing coagulation protein deficiencies
activation of coagulation factors to form a stabilizing fibrin clot, clot and important bleeding disorders. However, they can be misleading,
retraction to repair endothelial damage, and finally clot breakdown as they can also detect abnormalities of questionable clinical signifi-
through fibrinolysis (Fig. 129.1). Commonly available laboratory cance. The clinician must therefore understand the distinction
tests focus on the individual components of hemostasis by testing between the complexity of physiologic hemostasis and the simplistic
coagulation proteins, platelets, and fibrinolytic proteins; this chapter picture presented by laboratory tests.
is organized into similar components, to provide a structured labora- As we proceed in reviewing tests for coagulation proteins, we can
tory approach to the patient with a hemostatic or thrombotic place them into three technical categories:
problem. Yet the clinician must be mindful that in the body, the
components of hemostasis work together to form a product that is 1. Immunologic tests, which include enzyme-linked immunosor-
more than the sum of its parts. bent assays (ELISA), immunoelectrophoresis, and immunotur-
bidometric (latex agglutination) tests. These tests are quantitative,
and detect specific proteins with polyclonal or monoclonal anti-
LABORATORY EVALUATION OF COAGULATION PROTEINS bodies. Their sensitivity and specificity depend on the antibody
used (polyclonal versus monoclonal) and the presence of interfer-
ing substances (e.g., rheumatoid factor, other autoantibodies).
The Physiology Underlying Laboratory Evaluation of 2. Chromogenic or amidolytic assays, which measure the activity
Coagulation Proteins of the serine proteases of the coagulation system as they react
with synthetic peptides. The reaction (and thus the activity of
For over half a century, the process of fibrin clot formation has been serine protease) can be measured as the synthetic peptide releases
conceptualized as a “coagulation cascade.” This is based on the a colored dye. Chromogenic assays are affected by the specificity
waterfall hypothesis of Davie, Ratnoff, and MacFarlane, who almost of the peptide substrate. A disadvantage of these assays is that
simultaneously reported a sequence of proteolytic reactions starting their fairly narrow measure of an enzyme’s activity may not cor-
with factor XII (Hageman factor) activation by surface contact and relate with its biologic activity in vivo or its activity in clot-based
1,2
ending with thrombin’s proteolysis of fibrinogen to form fibrin. assays.
However, upon its introduction, this hypothesis of successive neces- 3. Clot-based or coagulation assays that are functional, and
sary proteolytic reactions already appeared to be too simplistic. Nearly compare the clotting potential of a patient’s plasma with standard
a decade earlier, Ratnoff had identified that deficiencies of factor XII plasma that has a known clotting potential. These tests are more
were not associated with bleeding; others soon established that defi- difficult and time-consuming to perform than the others, and are
ciency of factor XII’s cofactors (prekallikrein and high-molecular- susceptible to interference from other factors. However, they most
weight kininogen) did not result in a bleeding phenotype either. In closely approximate in vivo hemostasis.
1977, Osterud and Rappaport recognized that the factor VIIa/tissue
3
factor complex can activate factor IX to factor IXa. Broze later rec-
ognized that this complex cannot directly activate factor X, because SCREENING FOR COAGULATION PROTEIN DEFECTS:
of the presence of tissue factor pathway inhibitor (TFPI); factor IX
activation is a prerequisite. Though deficiency of factor XII and its ACTIVATED PARTIAL THROMBOPLASTIN TIME,
antecedents is not associated with bleeding, factor XI deficiency is. PROTHROMBIN TIME AND THROMBIN CLOTTING TIME
In 1991, Gailani and Broze explained this by proposing that factor
XI can be activated independent of factor XII, as formed thrombin The three assays most commonly used to screen for coagulation
cycles back to activate factor XI and thus amplifies its own forma- protein defects are: (1) the activated partial thromboplastin time
4
tion. Factor XII, long considered to have no role in coagulation in (APTT), induced in vitro by surface-activation of factor XII; (2) the
vivo, has been found to play a role in thrombus formation and prothrombin time (PT), induced in vitro by the addition of excess
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angiogenesis. Our understanding of hemostasis has advanced still tissue factor; and (3) the thrombin clotting time (TCT), a test of
further in recent years, as blood coagulation research is increasingly fibrinogen integrity and thrombin inhibition. The PT, APTT, and
performed under flow conditions with cellular elements in vitro, and TCT are clot-based assays that measure the rate of clot formation,
in live animals in vivo. and are based on the waterfall hypothesis of coagulation. They trigger
The physiologic “coagulation cascade” now appears to be an a sequence of proteolytic reactions in the intrinsic, extrinsic, and
intricate system with built-in shortcuts and feedback loops. It is common pathways (Fig. 129.3). The reactions culminate in the
triggered by factor VIIa and tissue factor, and results in the activation proteolysis of fibrinogen to form a fibrin clot, which causes soluble

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