GENERAL PRINCIPLES
The hemostatic system is a complex, regulated, sequence of reactions involving interactions among platelets, endothelium, and coagulation factors (Table 5-1).
DIAGNOSIS
Workup of suspected hemostasis disorders (Table 5-2; Fig.5-1)
Complete blood count (CBC)
Reveals thrombocytopenia if present.
Assesses whether the patient has developed clinically significant anemia
Leukocytosis or leukopenia may implicate a hematologic malignancy as the cause of a patient’s coagulopathy or thrombocytopenia.
Peripheral smear reveals the presence of microangiopathy, platelet clumping, and red and white blood cell morphology.
Prothrombin time/international normalized ratio (PT/INR)
Historically, PT values have varied from institution to institution due to differences in commercial thromboplastin. The INR system has markedly reduced interlaboratory variability. This ratio standardizes all PT assays and is calculated as
INR = [(PT patient)/(PT laboratory mean)]ISI
(ISI—International Sensitivity Index for the thromboplastin reagent used)
Activated partial thromboplastin time (aPTT)
Thrombin time (TT)
Workup of elevated PT or aPTT1
First, pre-analytical variables should be considered, such as
incomplete filling of blood collection tubes
heparin contamination
other confounding factors include a high hematocrit (>55%) and plasma turbidity (lipemic, hemolyzed, or icteric specimens)
Second, determine which pathway of the coagulation cascade is defective (see Table 5-2):
prolonged PT alone—extrinsic pathway
prolonged a PTT alone—intrinsic pathway
prolonged PTT and a PTT—both pathways
Third, perform a mixing study to determine whether the abnormality is due to an inhibitor present in the patient’s plasma or a factor deficiency (Figs. 5-2 through 5-4).
Mixing studies (Table 5-3)
Goal: to determine whether a prolonged PT or aPTT is due to factor deficiencies or inhibitors.
Method: patient’s plasma is mixed with normal plasma at a 1:1 ratio and the PT or the aPTT is performed immediately and after incubation at 37°C for 1 hour.
Workup of lupus anticoagulants (LAs)
LAs may occur in the presence or absence of systemic lupus erythematosus or other autoimmune diseases. They are associated with recurrent fetal loss and venous or arterial thromboembolic disease.
Routine aPTT and PT reagents are not sensitive enough to be used to screen for LA, so modifications have been made to develop LA-sensitive clotting tests with the following steps: (a) demonstration of prolonged phospholipid-dependent clotting time (such as modified aPTT, PT, or common pathway clotting test); (b) persistent prolongation of the clotting time after mixing with normal pooled plasma; (c) neutralization of the inhibitor by addition of excess phospholipid; (d) ruling out of a specific factor inhibitor (such as anti-factor VIII autoantibody). No LA test will detect 100% of LAs, therefore current guidelines require using at least two different sensitive clotting tests to screen for and confirm the presence of LA. If one or both tests are positive, testing should be repeated at least 12 weeks later. It is only clinically significant if positive LA is persistent and associated with an increased risk of future thrombotic complications.
FIGURE 5-1. The normal coagulation cascade is split into the intrinsic pathway and the extrinsic pathway, either of which leads to activation of factor X to Xa. The factors after that point are referred to as the common pathway.Disorders of the intrinsic pathway are manifest as prolongation of the aPTT. Disorders of the extrinsic pathway are reflected by prolongation of the PT, whereas disorders of the common pathway will prolong both tests.
Coagulation factor assays
These are clot-based assays to determine the functional levels of the factors in the coagulation cascade. These tests are usually obtained when there is a suspicion of factor deficiency or inhibitors.
Factor VIII assay
Goal is to determine factor VIII level.
FIGURE 5-2. General workup of an elevated PT in the setting of a normal aPTT.
Crucial test for diagnosing hemophilia A and von Willebrand disease (vWD) and for monitoring response to therapy in hemophilia.
Method: Determine the level of factor VIII in the patient’s plasma by mixing dilutions of the patient’s plasma with factor VIII–deficient plasma and comparing the result to that of similar dilutions of normal pooled plasma mixed with factor VIII–deficientplasma.
FIGURE 5-3. General workup of an elevated aPTT in the setting of a normal PT.
FIGURE 5-4. General workup of an elevation of both the PT and the aPTT.
Assessing deficiency of other factors
The same procedure as described for the factor VIII assay is used to determine the activity of other intrinsic pathway coagulation factors, using the corresponding deficient plasma for dilutions.
The extrinsic and common pathway factor activities are determined by comparing PT results for patient and normal pooled plasma diluted in plasma depleted of factor VII, X, or V or prothrombin.
Factor VIII inhibitor quantification (Bethesda units)
Indicated to test for factor VIII inhibitors. Factor VIII inhibitors are suspected when there is a normal PT; a prolonged aPTT that is partially corrected immediately after 1:1 mixing with normal plasma but prolonged again after 1 to 2 hours of incubation; and a very low factor VIII activity (usually <1%).
Appropriate treatment requires determining the titer of the inhibitor, which is measured as arbitrary Bethesda units.
Method: Serial dilutions of patient plasma are mixed with an equal volume of normal pooled plasma, and residual factor VIII activity is measured after 2 hours of incubation at 37°C.
The Bethesda titer is the reciprocal of the plasma dilution that neutralizes 50% of the factor VIII activity of normal pooled plasma.
A titer of BU <5 indicates a mild inhibitor that may be overwhelmed by larger infusions of factor VIII, whereas an inhibitor with a titer of BU >5 will likely require infusion of a bypass coagulation concentrate such as recombinant factor VIIa or activated prothrombin concentrate complex toachievehemostasis.
Studies of platelet function
Bleeding time
Method: Standardized incision is made in the patient’s forearm, and the duration of time to cessation of bleeding is measured.
Indication: To detect primary hemostasis defects (vWD and qualitative platelet disorders) in patients with histories of recurrent mucosal bleeding.
Although it can be a useful test for identifying problems with primary hemostasis, many variables may artificially prolong the bleeding time, such as medications that interfere with platelet function, differences in operator procedure and interpretation, subcutaneous edema, or thinning of the skin.
It is not an accurate predictor of surgical bleeding risk in asymptomatic subjects; therefore, its use has markedly declined and replaced by PFA-100.
Platelet function analyzer-100 (PFA-100)2
An in vitro screening test of primary hemostasis.
Method: Citrated whole blood is aspirated through a microscopic hole in a piece of nitrocellulose paper coated with collagen and epinephrine (COL/ EPI) or collagen and ADP (COL/ADP). The flow conditions mimic the shear stress in a capillary bed. vWF adheres to the collagen, and platelets adhere to the vWF, followed by agonist-mediated activation, aggregation, and eventual closure of the hole. The time to occlusion is reported as the closure time. The COL/EPI cartridge is sensitive to qualitative platelet disorders, especially aspirin effect, and vWD. The COL/ADP cartridge is less sensitive to aspirin but remains sensitive to vWD. Neither cartridge is sensitive to ADP receptor inhibitors such as clopidogrel (Plavix).
Normal PFA-100 closure times do not rule out mild qualitative platelet disorders, and if clinical suspicion is high, platelet aggregation studies should be performed.
PFA-100 has not been validated for preoperative assessment of bleeding risk in asymptomatic patients.
A hematocrit of <30% or a platelet count of <100,000/μL can produce false-positive results.
Platelet aggregation studies
Indicated when an inherited qualitative defect in platelet function is suggested by the clinical and/or family history.
Method: Various aggregating agents (arachidonic acid, collagen, ADP, epinephrine, and ristocetin) are added to the initially turbid platelet-rich plasma specimen. The platelets clump, permitting more light to pass through the plasma. The results with each agent are displayed graphically and interpreted as normal or abnormal.
This qualitative test is very labor intensive and should only be performed in highly selected cases.
As many prescription or over-the-counter medications can affect in vitro measurements of platelet function, almost all inpatients should be excluded.
Outpatients must discontinue aspirin-containing medications and clopidogrel (Plavix) for at least 7 days and NSAIDs for at least 72 hours before testingtoavoidfalse-positiveresults.
Laboratory evaluation of suspected von Willebrand disease (seeTable 5-4)3 Important tests to evaluate vWD include vWF antigen (vWF:Ag), factor VIII activity, ristocetin cofactor activity assay, collagen-binding assay, vWF multimer assay, ristocetin-induced platelet aggregation (RIPA) analysis, and factor VIII binding ELISA. No single test is adequate to diagnose vWD by itself. When abnormal tests are found suggesting vWD, these tests should be repeated to confirm the diagnosis. If two sets of tests do not agree, testing symptomatic first-degree relatives for vWD may be appropriate. See Chapter 7for detailed discussion of vWD.
von Willebrand factor antigen concentration (vWF:Ag)
vWF:Ag levels are measured against a normal reference sample and typically levels >50% of reference are considered normal.
Note that vWF:Ag reference intervals are blood type dependent. Healthy type O subjects may have vWF concentrations as low as 40% compared to normal pooled plasma, while the lower limit of the reference range for non-O controls will be ≥50%. Most laboratories do not provide blood type-specific reference intervals and are likely to have a lower limit ~50%. Therefore, some healthy individuals with blood type O and unimpressive personal and family histories of abnormal bleeding may have vWF:Ag, vWF activity, or factor VIII activity <50% due to their blood type but are incorrectly diagnosed with type 1 vWD.
The vWF:Ag concentration will be abnormally low in all type 3, most type 1, and some type 2 vWD.
Ristocetin cofactor assay
The ability of the patient’s plasma (platelet poor plasma) to aggregate normal platelets in the presence of ristocetin is compared with that of a normal pooled plasma specimen.
Ristocetin is an antibiotic that that was off the market because it causes severe thrombocytopenia due to in vivo agglutination of platelets to vWF.
More than 50% is considered normal, but healthy blood type O patients may have lower activity (see discussion above).
This test is useful when used with the vWF:Ag test in the vWF ristocetin cofactor/vWF:Ag ratio. A ratio ≤0.7 suggests a qualitative defect of vWF, which indicates a diagnosis of vWD type 2A, 2B, or 2M and requires vWF multimer assay and RIPA analysis for finaltyping.
Collagen-binding assay
This ELISA is especially useful for identifying the presence of high molecular weight forms of vWF. Collagen is immobilized in the test well, and following incubation with plasma and washing, bound vWF is measured.
Patients with types 2A and 2B vWD are deficient in high molecular weight vWF, therefore this test is useful in their diagnosis. As described earlier, reference comparison with >50% levels are considered normal.
This test is used in comparison with the vWF:Ag test in the vWF collagen binding/vWF:Ag ratio (normal, >0.7).
This test is not popular and rarely used in the United States.
von Willebrand factor multimer assay
The multimer assay involves labor-intensive gel electrophoresis techniques to separate vWF into bands that normally range in size from 0.5 to 20 million daltons.
Loss of large and intermediate multimers is seen in type 2A and 2B vWD.
Normal distribution of vWF multimers is seen in type 2M cWD.
This should not be part of initial workup for vWD, only indicated for suspected type 2A/2B/2M, possibly type 3.
This test is not routinely performed at most institutions.
Ristocetin-induced platelet aggregation (RIPA) analysis (Table 5-5)
The aggregation of the patient’s platelets is tested in the presence of different concentrations of ristocetin.
Used for differentiating vWD type 2B from 2A or 2M.
RIPA is used only after the diagnosis of vWD is made and further clarification of subtype is necessary.
Factor VIII binding ELISA
Indicated to assist in the diagnose type 2N vWD, which is characterized by impaired binding of vWF to factor VIII.
PFA-100 closure time
Can be used to screen for vWD in patients with an appropriate bleeding history, and to monitor response to 1-desamino-8-D-arginine vasopressin (DDAVP; which stimulates release of vWF stored in endothelial cells) or infusion of vWF concentrate (Humate-P), since other vWF tests may not be available on a STAT basis.
REFERENCES
1. Kamal AH, Tefferi A, Pruthi RK. How to interpret and pursue an abnormal prothrombin time, activated partial thromboplastin time, and bleeding time in adults. Mayo Clin Proc. 2007;82:864–873.
2. Hayward CP, Harrison P, Cattaneo M, et al. Platelet function analyzer (PFA)-100 closure time in the evaluation of platelet disorders and platelet function. J Thromb Haemost. 2006; 4:312–319.
3. Sadler JE, Budde U, Eikenboom JC, et al. Update on the pathophysiology and classification of von Willebrand disease: a report of the Subcommittee on von Willebrand Factor. J Thromb Haemost.2006;4:2103–2114.