Coagulation Testing in the Core Laboratory

William E. Winter, MD; Sherri D. Flax, MD; Neil S. Harris, MD

Disclosures

Lab Med. 2017;48(4):295-313. 

In This Article

Special Coagulation Studies

Mixing Studies: The 1:1 Mix

The mixing study assists in the assessment of an abnormally prolonged PT or aPTT. An equal volume of citrated patient plasma is mixed with normal pooled plasma (NPP), and the PT or aPTT are repeated on the 1:1 mix. If the assay time corrects (ie, the clotting time is now within PT or aPTT reference interval), the prolongation is likely due to either single or multiple clotting factor deficiencies or dysfunctions. In contrast, the presence of inhibitors in the specimen will result in a lack of correction following mixing of the patient plasma and the NPP. Even if the result decreases significantly, but the clotting time is still above the reference interval, it is deemed a lack of correction. Inhibitors include heparin, direct-acting oral anticoagulants and autoantibodies directed against coagulation factors or autoantibodies directed against phospholipid-binding proteins such as occurs in the antiphospholipid syndrome (APLS).

Factor Activity Assays

These assays are most commonly performed as a 1-stage assay. An essential component of the assay is a factor-deficient substrate plasma that lacks the specific factor being evaluated.

  1. The patient's citrated plasma is diluted 1:10, 1:20, and 1:40. The diluted specimen is then mixed 1 to 1 with the factor-deficient substrate plasma. The patient's specimen therefore supplies the missing factor.

  2. A PT or aPTT is performed on the above mix, depending on the factor being tested. For testing factors VIII and IX, for example, one would run an aPTT (intrinsic pathway and common pathway), while for a FVII assay, a PT (extrinsic pathway and common pathway) would be performed.

  3. The assay is calibrated using a standard reference plasma with a known concentration of the factor being evaluated.

  4. A standard curve is plotted, with the factor concentrations on the x-axis and the aPTT (or PT) clotting times on the y-axis. The x- and y-axes are usually displayed as a semilog plot.

  5. The standard curve is used to interpolate or translate the clotting times of the unknown specimen into specific factor concentrations.

Factor Inhibitors

Factor inhibitors are most commonly diagnosed in male patients with severe hemophilia A (FVIII deficiency) where the inhibitors occur as a result of factor replacement therapy. The prevalence of FVIII inhibitors is about 10% to 15% in such patients. These inhibitors are alloantibodies that result from exposure to human FVIII in a setting of an absence or near absence of endogenous FVIII. These antibodies produce resistance to the replacement factor and may manifest as bleeding, despite adequate dosing of the factor.

Factor inhibitors can also appear in the form of spontaneous autoantibodies in individuals who were previously well and had no prior bleeding problems. This is an autoimmune condition called acquired hemophilia. It is seen in both men and women who are often middle aged or older. Acquired hemophilia is associated with very severe bleeding, usually within skin, soft tissue, muscles, and the gastrointestinal tract. Hemarthroses (seen in congenital hemophilia) are rare. Although FVIII activity is low, the bleeding often appears out of proportion to the reduced FVIII level. There is some association with other autoimmune diseases, as well as with lymphoproliferative disorders and solid tumors. However, such associations are not seen in every case.

If a FVIII inhibitor is present, the aPTT will be prolonged with a normal PT, and FVIII activity will be low, especially in acquired hemophila. The TT is normal as is the dilute Russell viper venom time (dRVVT). The 1:1 aPTT mix either shows no correction, or initially corrects, but then the clotting time prolongs after incubation for 1 to 2 hours at 37°C. This later prolongation is the result of catalytic antibodies that degrade FVIII.

It is important to measure the titer (concentration) of the inhibitor. This is achieved by incubating the diluted patient plasma with NPP for two hours at 37°C (Figure 18). At the end of the incubation, the residual FVIII activity in the mix is determined by comparing it with a similarly diluted and incubated control specimen. If the titer of the inhibitor is 1 Bethesda unit (1 BU), the residual factor activity is 50%; at 2 BU, 25% activity remains; and 12.5% residual activity means that the titer is 3 BU. A titer less than 5 BU indicates that the inhibitor can be overcome by the administration of additional FVIII. For higher titers (≥5 BU), this therapy is no longer effective. At this point, therapy includes some type of bypassing agent. This may be either recombinant FVIIa (Novoseven RT, Novo Nordisk Inc, Plainsboro, New Jersey) or a prothrombin complex concentrate (PCC). The latter is a 4-factor preparation. Factors II, IX, and X in the PCC preparation are mainly nonactivated, while factor VII is mostly in the activated form. For adults with acquired hemophilia A, an alternative therapy is a recombinant porcine Factor VIII (Obizur, Baxalta Incorporated, Bannockburn, Illinois). In addition to these alternative FVIII therapies or bypassing agents, an important treatment is the introduction of immunosuppression to reduce the antibody response against FVIII.

Figure 18.

This is the basic setup for a Bethesda Inhibitor titer assay. Dilutions of the patient specimen in buffer are shown on the left. These samples are mixed with an equal volume of normal pooled plasma. The mix is incubated at 37°C for 2 hours, and the residual factor activity is determined relative to a control. The control (not shown) is made by mixing the diluting buffer with an equal volume of normal plasma, and this is also incubated at 37°C for 2 hours.

Specific Tests for Von Willebrand Disease

In von Willebrand disease (VWD), there is defective synthesis or release of functional von Willebrand factor (VWF), which results in defective primary hemostasis. The condition can be associated with decreased antigenic and functional activity (Types 1 and 3) or with a relatively normal antigen concentration in the setting of reduced functional activity (Type 2).

Most coagulation laboratories can measure the plasma concentration of VWF protein (VWF antigen) by an immunoturbidimetric technique. Testing the functional activity of VWF utilizes the drug ristocetin. Ristocetin facilitates receptor (GPIb) and ligand (VWF) interaction in this agglutination (GPIIb-IIIa independent) reaction. VWF activity is determined by using patient's plasma (the source of VWF) in combination with formalin-fixed lyophilized platelets (the source of GPIb) and ristocetin. This procedure is called the ristocetin cofactor activity. This is in contrast to ristocetin-induced platelet aggregation (RIPA), which utilizes living patient-derived platelets and patient plasma. The latter test is used in rare variants (Type 2B) that produce an unusually brisk aggregation response in the presence of low concentrations of ristocetin.

The state of multimerization of VWF is important and is assessed by electrophoresis on agarose gels (Figure 19). Normal plasma produces an extended "ladder" of multimers including high molecular weight forms. Type 2A and 2B VWD are associated with the lack of intermediate and high molecular weight multimers.

Figure 19.

Schematic representation of von Willebrand factor (VWF) multimers as illustrated by western blotting and VWF detection with VWF antibodies. The distribution of VWF multimers is assessed by agarose gel electrophoresis, which separates the multimers according to their molecular weight, with the larger multimers being at the top of the figure. The western blot on the right represents the loss of those multimers that are essential to normal VWF function.

von Willebrand antigen concentrations and/or activity assays should be performed in tandem. When interpreting results, one must be aware that individuals with blood group O have the lowest mean VWF antigen levels as compared to other blood groups (AB has the highest mean level). Other ancillary tests include a FVIII activity determination because low VWF is frequently associated with a low FVIII activity (VWF is the carrier protein for FVIII). If the FVIII activity is sufficiently decreased, the aPTT may be prolonged.

In summary, the laboratory workup for VWD should include the following evaluations (von Willebrand Disease. Leebeek FW, Eikenboom JC):

  1. Platelet count

  2. Platelet function analyzer-100 (PFA-100 or equivalent), which tests the ability of platelets in flowing blood to obstruct an aperture in a collagen-coated cartridge

  3. aPTT

  4. Factor VIII activity

  5. VWF antigen determination

  6. VWF activity determination (ristocetin cofactor activity or additional collagen-binding activity)

  7. VWF multimer analysis

The RIPA may be used in very selected cases where Type 2B VWD is suspected.

The Anti-Xa Assay

Heparin activity in plasma can be directly assessed by a chromogenic procedure commonly referred to as an anti-Xa assay (Figure 3). Generally, the reaction mixture contains exogenous FXa as well as a chromogenic substrate for FXa. Some versions of the assay utilize the patient's own AT, while others supplement with exogenous AT. Irrespective, in both methods, heparin, present in the specimen, complexes with AT, and this complex inhibits FXa. Any residual FXa cleaves the synthetic chromogenic substrate, releasing a yellow-colored chromophore (p-nitroaniline), which is read optically at 405 nm. The amount of chromophore released is inversely proportional to the concentration of heparin present. Results are expressed as units per mL of anti-Xa activity. The assay can be automated and can be used to measure both unfractionated heparin and low molecular weight heparin, as well as fondaparinux when properly calibrated.

Testing for the Antiphospholipid Syndrome

The APLS is an acquired autoimmune phenomenon associated with an increased incidence of both venous and arterial thromboses, as well as fetal loss or premature birth. The diagnosis of this syndrome requires that both clinical signs and laboratory features be met. Typically, there is a paradoxical prolongation of the aPTT in the absence of any clinical features of bleeding. This is the so-called lupus anticoagulant (LA) effect. Most often, the prolonged aPTT does not correct on a 1:1 mix; however, it is not that uncommon to encounter correction in the presence of a weak LA.

The laboratory definition of the APLS requires the presence of either an LA or a persistent titer of antiphospholipid antibodies. Persistent is defined as repeatedly positivity after a minimum of 12 weeks interval between tests.

Lupus Anticoagulant Testing

One of the more commonly used LA assays is the dilute Russell viper venom time (dRVVT) in which the common coagulation pathway is activated through the action of a snake venom that converts FX to FXa. The advantage of the dRVVT is that it is not affected by hemophilia or by antibodies to factors VIII and IX or by an elevated FVIII level.

The dRVVT assay requires an initial baseline reaction (referred to as screening) followed by a confirmatory step in which the reaction is supplemented by exogenous phospholipid, revealing correction of the previously prolonged aPTT clotting time. A true LA effect is characterized by shortening of the clotting time on phospholipid supplementation, while factor deficiencies are impervious to this step. Many dRVVT reagents contain a cationic heparin neutralizer that binds and inactivates unfractionated heparin when present in the therapeutic range.

In order to assess if the clotting time of the confirmatory step is shortened relative to the screening step, it is common practice to establish a ratio of screening-to-confirm times. These ratios are typically accepted as positive for a lupus anticoagulant effect if they are greater than 1.2. Recently, this approach has been modified by several national and international organizations. The important modification is that both the screening and confirm times themselves be converted to normalized ratios by dividing the dilute Russell viper venom clotting time of the unmodified patient specimen by that of normal plasma.

Antiphospholipid antibodies are those antibodies that interact with phospholipid-binding proteins or phospholipids themselves (eg, cardiolipin, which is present primarily in mitochondrial inner membrane). International guidelines recommend detection of either IgG or IgM antibodies to cardiolipin (aCL) or to beta-2 glycoprotein I (aβ2GP1). Antiphospholipid antibodies are identified by ELISA. The specimen from the patient is added to the ELISA well, and if the appropriate antibodies are present, they will bind to the target antigen coating the well. The laboratory definition of antiphospholipid syndrome requires that a significant titer of aCL or aβ2GP1 persist for at least 12 weeks. International guidelines recommend that positive antibody titers are titers above the 99th percentile of the population reference interval.

Direct-Acting and Novel Anticoagulants

The direct-acting novel, oral anticoagulants (DOACs or NOACs) are intended to replace warfarin as orally administered agents. There are two broad classes of DOACs (Figure 21): 1) the oral direct thrombin inhibitors (DTIs), such as dabigatran; and 2) the oral direct factor Xa inhibitors, such as rivaroxaban and apixaban. Therapeutic monitoring is usually not necessary; however, in certain circumstances monitoring becomes vital, including in patients with renal impairment, patients with significant bleeding, patients with extremes of body weight, and when there is apparent treatment failure.

Figure 21.

On the left, is the factor Xa/Va prothrombinase complex. Direct Xa inhibitors block the action of FXa without the need for antithrombin. Direct factor Xa inhibitors are able to inhibit free, prothrombinase-bound (as shown here), and clot-associated FXa. Thrombin (FIIa) is shown on the right. The direct thrombin inhibitors (DTIs) inhibit thrombin without the need for antithrombin.

There are 2 broad groups of laboratory assays: 1) quantitative assays designed to monitor plasma concentrations of the drug; and 2) qualitative, or noncalibrated, assays, which can indicate if the drug is present or absent in a plasma sample.

All of these direct-acting medications can be measured quantitatively by liquid chromatography/mass spectroscopy. Such analytical methods are time consuming and not easily transferrable to the majority of clinical diagnostic laboratories. Furthermore, quantitative therapeutic ranges are not well established. Quantitative measurements of the direct Xa inhibitors are possible using anti-Xa chromogenic assays calibrated with either rivaroxaban or apixaban. For the direct thrombin inhibitors, the most practical quantitative assay is the dilute TT. This differs from the standard TT because the specimen is diluted 1:4 in normal plasma prior to being assayed.

Qualitatitive, or noncalibrated, assays are best used for their negative predictive value. Firstly, for dabigatran there is the standard TT measurement. A normal TT can be used to exclude the presence of an oral direct thrombin inhibitor. For rivaroxaban and apixiban, one can use a heparin-calibrated anti-Xa assay designed for measuring either unfractionated or low molecular weight heparin. An anti-Xa value of less than 0.1 u/mL excludes the presence of rivaroxaban and apixaban.

The PT and aPTT are variably affected by the NOACs and generally unhelpful in monitoring their concentrations. Most importantly, a normal PT or aPTT does not exclude the presence of any of the NOACs. In general, dabigatran prolongs the aPTT more than the PT; the opposite is true for rivaroxaban, where the PT is the test that is mostly prolonged; however, this depends on the PT reagent in use.

Summary

Appropriate test ordering and interpretation are based on a thorough understanding of normal and pathologic coagulation, and the similarities and differences between in vivo and in vitro coagulation. The basic tests used to assess coagulation include platelet count, PT, and aPTT. More sophisticated testing (Figure 20) is then undertaken depending on the results of these antecedent tests guided by the patient's initial assessment consisting of the patient's history and physical examination.

Figure 20.

An outline of the anti-Xa assay which can be adapted to measure plasma concentrations of heparin, including unfractionated heparin, low molecular weight heparin, and fondaparinux, an ultra-low molecular weight synthetic heparin. The basis for the assay is the cleavage of a synthetic peptide by exogenous (ie, not from the specimen) FXa. This cleavage releases a colored product. In the presence of heparin and antithrombin, the exogenous Xa is inhibited, as is the color development.

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