Page 2175 - Hematology_ Basic Principles and Practice ( PDFDrive )

1928 Part XII Hemostasis and Thrombosis

mucocutaneous bleeding. In the past, the Ivy bleeding time—a test are to the added agonist. The increase in light transmission can be
in which the forearm is cut, and the time until bleeding stops is measured spectrophotometrically. Recommended agonists include
measured—was used to screen for disorders of primary hemostasis, ADP, epinephrine, arachidonic acid, collagen, and thromboxane
14
17
including platelet function disorders (PFDs) and vWD. This test analogue U46619 at predetermined concentrations. Ristocetin, an
is no longer considered to be useful in the investigation of bleed- antibiotic that facilitates vWF binding to the glycoprotein Ib/IX/V
ing disorders. It is invasive, operator dependent, can be affected by complex, is also used in LTA to cause platelet agglutination. ADP
nonhematologic factors (e.g., Ehlers-Danlos syndrome, osteogenesis and epinephrine result in a biphasic aggregation pattern; primary
imperfecta, scurvy, skin quality, skin temperature, vascular function), aggregation occurs in response to activation of the platelet glycopro-
15
and has low predictive value for surgical bleeding. It is also not tein IIb/IIIa receptor, then platelets degranulate and cause secondary
useful in detecting platelet dysfunction in the setting of thrombocyto- aggregation. Conversely, arachidonic acid, thromboxane analogue,
penia. Importantly, the bleeding time is neither sensitive nor specific and collagen result in a monophasic aggregation pattern. Several
18
enough to preclude further testing for vWF or platelet abnormali- defects of platelet function have well-characterized LTA patterns.
16
ties. A normal bleeding time should not reassure the clinician that Bernard-Soulier syndrome, a defect in glycoprotein Ib, results in
an important bleeding disorder is not present. normal aggregation to ADP and collagen, but absent agglutination
Several automated analyzers have been proposed as replacements to ristocetin (this pattern can also be seen in vWD.) Glanzmann
for the bleeding time, and potentially useful screening tools for dis- thrombasthenia, a defect in glycoprotein IIb/IIIa, results in an absent
orders of primary hemostasis. The platelet function analyzer PFA-100 primary response to ADP and collagen. Impaired aggregation to
(Siemens, Deerfield, IL), has been best evaluated. The PFA-100 arachidonic acid suggests an aspirin effect, or cyclooxygenase defi-
attempts to recreate the high shear conditions under which primary ciency. Platelet storage pool disorders can be diagnosed with LTA and
hemostasis occurs in vivo. Using citrated whole blood, it measures studies that measure proteins within or released from platelet
the closure time required for platelets to adhere to agonist-coated α-granules or dense granules. Tests of ATP secretion from platelets
membranes and aggregate under high shear stress (5000–6000/s). can also be valuable in assessing PFDs, as they complement and
19
Two cartridges are available: one with collagen–adenosine diphos- expand on LTA results. ATP secreted by dense granules is measured
phate (ADP) membranes, and the other and collagen–epinephrine by a sensitive luminescent (firefly luciferin-luciferase) assay for extra-
coated membranes. Platelet function is measured as a function of the cellular ATP. Platelet electron microscopy is increasingly used to
time needed to occlude the aperture, termed closure time. Abnormali- evaluate the ultrastructure of platelets, including the dense granules.
ties can lead to lengthening of the closure time. The PFA-100 is more Finally, with appropriate monoclonal antibodies, flow cytometry can
sensitive than the bleeding time in patients with previously diagnosed examine the surface of the platelet. Flow cytometry has a variety of
vWD, but less sensitive in those with mild platelet secretion disorders uses, including detection of platelet activation by using antibodies to
(which are the most frequent PFDs) and platelet storage pool defi- proteins expressed on the platelet surface (e.g., P-selectin, thrombo-
ciency. In a prospective study by Quiroga et al, the overall sensitivity spondin), diagnosis of platelet surface glycoprotein deficiencies, and
16
of closure time in vWD and PFDs was 50% and 18%, respectively. measurement of dense granules.
This suggests that the PFA-100 is only marginally more useful than
the bleeding time. It is still not sensitive or specific enough to preclude
further testing for vWF or platelet abnormalities, and cannot defini- Evaluation of Von Willebrand Factor
tively rule out important bleeding disorders.
At this time, laboratory screening tests for disorders of primary vWD is discussed in detail in Chapter 138. It is diagnosed when
hemostasis have limited usefulness. Diagnosis of these disorders must patients have a history of mucocutaneous bleeding (usually with a
be grounded in a thorough clinical history and physical exam, and positive family history), and quantitative or qualitative abnormalities
ultimately relies on the performance of the specific and sensitive tests of vWF. Laboratory testing of vWD includes the factor VIII assay,
outlined in the following. vWF antigen (vWF:Ag), and vWF activity (most commonly mea-
20
sured with the ristocetin cofactor assay, vWF:Rco). Many laborato-
ries also test vWF further with the collagen binding assay (vWF:CB,
Evaluation of Platelets abnormal when there is a loss of high-molecular-weight vWF multi-
mers or impaired binding of vWF to vascular endothelium), the
The first step in the investigation of platelet disorders is determining low-dose ristocetin-induced platelet aggregation assay (abnormal in
the platelet count, as thrombocytopenia alone may increase bleeding loss-of-function and gain-of-function defects that characterize type
risk (thrombocytopenia is discussed in detail in Chapters 131 and 2B vWD and platelet-type pseudo-vWD), and vWF multimer analy-
20
132). Obtaining a detailed list of medications the patient is taking, sis (which is useful in type 2A and 2M vWD). Assays that measure
allopathic and naturopathic, prescription and over-the-counter, is factor VIII binding to vWF can identify patients with defects in
20
also essential, as many drugs impact platelet number and function. factor VIII binding, such as type 2N vWD. Highly specialized
Platelet function can also be altered in the setting of systemic disor- coagulation laboratories may also perform vWF gene sequence analy-
ders, such as renal disease, liver disease, myeloproliferative and sis and vWF propeptide analysis; the latter shows promise in detecting
myelodysplastic disorders, and malignancy. Inherited platelet disor- defects associated with accelerated vWF clearance. 20
ders are often syndromic, and are associated with manifestations like The diagnosis of vWD—particularly type 1 vWD, which is
albinism, bone changes, sensorineural hearing loss, renal disease, and defined as a partial, quantitative deficiency of vWF—can be challeng-
14
immunodeficiency. ing. Studies have shown a high prevalence of bleeding symptoms in
Platelet function can be evaluated with platelet aggregation normal, healthy individuals. By definition, 2.5% of the population
studies. These tests measure the ability of agonists to cause in vitro will also have a level of vWF:Ag that falls below the lower limit
platelet activation and aggregation. Two forms of platelet aggregation of laboratories’ normal reference range. Thus, there is a danger of
studies are available: light transmission aggregometry (LTA) in coincidentally associating low vWF with bleeding symptoms, and
21
platelet-rich plasma and impedance aggregometry in platelet-rich overdiagnosing type 1 vWD in the general population. To further
plasma or whole blood. LTA in platelet-rich plasma is considered the complicate the situation, the normal range of plasma vWF is quite
gold standard of platelet function testing, whereas impedance broad. vWF is an acute phase reactant, and can go up in patients
aggregometry is less well characterized. A number of recent guidelines who are inflamed, infected, pregnant, under stress, or suffering
17
have laid out standards on how to perform and interpret LTA. from comorbid diseases like cancer. vWF goes up with age, and is
Briefly, when a platelet agonist is added to platelet-rich plasma, lower in individuals with blood type O. It is recommended that the
aggregation occurs and light transmission increases. When factors like label of type 1 vWD be applied only when the clinical presenta-
temperature, platelet count, and mixing speed are controlled, the tion is consistent, and when vWF levels are persistently low and
amount and rate of this increase depends on how reactive platelets <30 IU/dL. 14