December Best Practice in Platelet Function Testing Stan McCormick, MD

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1 December 2012 Best Practice in Platelet Function Testing Stan McCormick, MD The indications for platelet function testing have expanded in recent years as improved testing technologies have become available in the clinical laboratories at Allina Health. In addition to patients with bleeding disorders, a variety of clinical settings have emerged for which platelet function assessment plays a critical role in patient management, such as the detection of P2Y12 inhibitor resistance in stroke and cardiac patients, or the pre-operative assessment of patients undergoing surgical procedures associated with a high-risk of bleeding. In patients with unexplained bleeding the work up often includes screening assays for Von Willebrand disease, which can mimic primary platelet dysfunction clinically. Three unrelated disorders share the seemingly paradoxical association of thrombocytopenia and thrombosis: (1) Patients undergoing unfractionated heparin anticoagulation, who may form plateletactivating antibodies (heparin-induced thrombocytopenia; HIT) and who are at risk for arterial and venous thrombosis; (2) Patients with paroxysmal nocturnal hemoglobinuria (PNH), for whom venous thrombosis represents a major cause of morbidity and mortality, and (3) Patients with thrombotic thrombocytopenic purpura (TTP), who are at risk for life-threatening microvascular thrombosis in one or more organs. In the first two of these thrombocytopenic/thrombotic disorders the laboratory plays an important role, whereas in the third (TTP) the diagnosis remains primarily clinical. In this article we focus on laboratory services routinely available within Allina Health that physicians may use in the daily management of patients with platelet -related clinical problems, mentioning only briefly some of the more esoteric send-out services available at referral centers.

2 Tests Available at Allina Health Laboratory for Assessment of Platelet Function Platelets are tiny (2uM) anucleate blood cells that promote hemostasis through 4 crucial functions: (1) Adherence to exposed tissue at sites of injury through specific receptors that bind collagen, fibrinogen, fibrin, and VWF. (2) Release of ADP and other agonists from platelet granules that accelerates platelet plug formation, (3) activation of GPIIb/IIIa receptors, leading to aggregation and formation of a hemostatic platelet plug, and (4) formation of a phospholipid substrate for the activation of coagulation factors. In one form or another, these 4 functions underlie most of the qualitative tests of platelet function available in the clinical laboratory, while state-of-the-art hematology analyzers provide several additional quantitative platelet parameters. Platelet Count and Platelet Indices Electronic enumeration of peripheral blood platelets with several clinically useful platelet indices is available in all Allina Health metro hospitals and clinics. Platelet Count (484/PLT) The Allina Health Laboratory reference interval for platelet count is thou/cumm, although adequate hemostasis may be observed with counts as low as thou/cumm, depending on patient-specific variables. The Allina Transfusion Care Committee (ATCC) has adopted specific platelet count thresholds for the therapeutic transfusion of platelet concentrates in most clinical situations. Despite the high level of accuracy and precision available with modern hematology analyzers, the accuracy of platelet counting may be limited by some common artifacts, such as the presence of clots in the sample, or when the patient s blood contains cold-reacting platelet agglutinins that produce platelet clumping in EDTAanticoagulated blood, leading to pseudothrombocytopenia. The presence of clots in the sample or platelet clumping in the blood smear invalidates the automated platelet count, which is not reported, although a smear estimate is provided. In these situations the laboratory may request a re-draw or a blood sample drawn into a sodium citrate (blue top) tube. Platelet Indices: Mean Platelet Volume (MPV) Ref Range: fl Platelet size, as measured in the hematology analyzer (MPV) or estimated microscopically from the blood smear (normal 2-4 um), is a clinically useful parameter. Newly produced platelets are larger than circulating platelets, which become smaller over the 7-10 day platelet lifespan. As a result, in patients with thrombocytopenia secondary to peripheral destruction the MPV is increased, reflecting active bone marrow compensation with release of young, leftshifted platelets. Patients with physiologic thrombocythemias associated with iron deficiency, acute infections, or bleeding most often have platelets of normal or even reduced size, as reflected in either the MPV or blood smear. In contrast, the abnormal thrombocythemias associated with chronic myeloproliferative neoplasms are characterized by platelets that are inappropriately large, resulting in an abnormally excessive total platelet mass. Thus, the finding of enlarged platelets in the blood smear of a patient with thrombocythemia, particularly if additional abnormalities of platelet granulation are seen, is a strong indication of a potential underlying primary bone marrow disorder. 2

3 Immature Platelet Fraction (Reticulated) (8931/IPF) Ref Range: % The percentage of newly produced platelets (Immature Platelet Fraction; IPF) in a blood sample is a clinically useful indicator of thrombopoiesis. The IPF is a measure of platelet immaturity analogous to reticulocyte count, and is determined simultaneously with platelet count and MPV in the hematology analyzer. The IPF has diagnostic value in patients with thrombocytopenia, and is useful in guiding platelet transfusion therapy following chemotherapy or during recovery from bone marrow aplasia. The IPF is increased in peripheral destructive thrombocytopenias, reflecting active bone marrow compensation with production of more immature platelets; whereas in the hypoproliferative thrombocytopenias the IPF is normal or decreased. Following chemotherapy the IPF increases as bone marrow regeneration begins, then subsequently declines as the platelet count begins to rise. Thus, in a patient with thrombocytopenia secondary to chemotherapy, a rising IPF signals the beginning of bone marrow regeneration, and platelet transfuson can be deferred, while a stable IPF suggests thrombopoiesis is still inadequate and platelet transfusions may be appropriate. Assessment of Platelet Morphology in the Blood Smear Assessment of platelet morphology in the blood smear can provide valuable qualitative information in the work up of patients with suspected platelet-associated bleeding. Normal platelet morphology is most often observed in patients with the peripheral destructive thrombocytopenias (such as ITP), benign physiologic thrombocythemias, and in patients with von Willebrand disease who present with bleeding disorders mimicking platelet dysfunction. The presence of red cell fragments implicates intravascular hemolysis as the mechanism of thrombocytopenia in patients with microangiopathic disorders (TTP, HELLP, HUS, DIC). In evaluating a patient with a potential heritable platelet disorder a request for blood smear morphology is an appropriate first step. Patients with Glanzmann thrombasthenia will have normal appearing platelets (and normal platelet count), whereas giant platelets with variable platelet counts are seen in Bernard-Soulier disease. Patients with Wiskott-Aldrich syndrome typically have small platelets. In the gray platelet syndrome (alpha granule deficiency), platelets appear pale, gray, and hypogranular; however, in some storage pool disorders (SPDs) platelet morphology may be normal by light microscopy, but abnormal (decreased alpha and/or dense granules) by electron microscopy. In the May-Heggelin syndrome platelets are enlarged and may approach the size of red blood cells, while leucocytes have prominent inclusions. Patients with myelodysplastic syndromes and myeloproliferative neoplasms frequently have qualitative platelet abnormalities independent of the platelet count that are detectable in the blood smear (Images 1-4). Image 1: Blood smear with morphologically normal platelets 3 Image 2: Platelets in iron deficiency anemia are normal to small in size and normally granulated

4 Image 3: A giant platelent with abnormal granulation in a patient with MDS Image 4: Enlarged and hypogranular platelets in a patient with Polycythemia Vera Platelet Function Testing (PFT/6950) Ref Range (CT): CEPI sec; CADP sec In 2005 platelet function screening with the bleeding time test was discontinued in favor of the more objective and reproducible PFA-100 point-of-care device, currently available in all Allina Health metro hospital laboratories. The PFT provides a global assessment of platelet adhesion and aggregation in whole blood. Blood is aspirated through a capillary tube in a test cartridge coated with either collagen + epinephrine (CEPI), or collagen + ADP (CADP). Under conditions of high shear normal platelets form a plug that stops flow in the test cartridge. The resulting closure time (CT) in seconds reflects overall platelet function. A normal CT indicates normal global platelet function, while a prolonged CT may be seen in several situations, including thrombocytopenia (<150,000/cumm), anemia (HCT <35%), and in several clinically relevant conditions (Figure 1). CEPI is the primary screening cartridge and will detect platelet dysfunction secondary to intrinsic platelet defects, most cases of von Willebrand disease, or platelet-inhibiting agents. Aspirin-like drugs cause a prolonged CEPI and a normal CADP, whereas intrinsic platelet dysfunction, von Willebrand disease, and non-aspirin drugs are typically abnormal with both cartridges. One important caveat: Owing to the high concentration of ADP in the CADP cartridge, the PFT test is not sensitive to clopidogrel and other anti-p2y12 inhibitors (see below). Whether the PFA-100 (PFT) is an adequate approach for the detection of aspirin resistance in stroke patients is not well established. Figure 1 4

5 Tests for Diagnosing Heparin-Induced Thrombocytopemia (HIT) Polyspecific (8775/PHA): IgG/IgA/IgM IgG Specific (8771/GHA): IgG Ref Range for each: Negative The paradoxical association of thrombosis in a patient with thrombocytopenia is a cardinal feature of the clinical syndrome, heparin-induced thrombocytopenia (HIT). The basis of HIT is an immune reaction resulting in antibodies formed against the platelet factor 4/heparin complex (PF4/H). No other platelet-related testing requires the level of integration of clinical and laboratory variables as those used in evaluating patients with suspected HIT. Effective use of the PHA/GHA assays requires awareness of three important caveats: (1) the formation of anti-pf4/h antibodies is very common in patients exposed to unfractionated heparin (UFH) (up to 50% of cardiac surgery patients), although only a small subset (~10%) of seropositive patients have antibodies capable of inducing platelet activation leading to thrombocytopenia and thrombosis. Nevertheless, in patients who develop true clinical HIT the diagnosis becomes urgent, as catastrophic sequelae may develop rapidly if UFH is continued. (2) The likelihood that a positive PHA/GHA reflects clinical HIT is heavily dependent on the pre-test probability, as demonstrated by one mnemonic, the 4T s score : defined by the level of T hrombocytopenia, T ime in number of days following initiation of heparin, presence of T hrombosis or other HIT-related sequelae, and lack of ot her explanations. A low 4T s score has high negative-predictive value, ruling out HIT in most situations (see references), and (3) In a positive sample, the optical density (OD, the endpoint of the PHA/GHA ELISA test) reflects the strength and titer of the heparin-associated antibodies and can enhance the specificity of the test (see below). Allina Health Laboratory offers daily heparin antibody testing using the polyspecific (Heparin Antibodies, Polyspecific; PHA) and IgG specific (Heparin Antibodies, IgG Specific; GHA) assays. The GHA assay is preferred in most situations as clinically significant heparin antibodies are nearly always of the IgG variety, whereas the polyspecific test (PHA) detects IgG as well as IgA and IgM antibodies, the latter being most often clinically irrelevant. Thus, the PHA is highly sensitive to the presence of anti-pf4/h antibodies (98%), but is less specific for the clinical syndrome of HIT (89%). The PHA test is useful in a patient with a low to moderate pre-test clinical likelihood of HIT, where a negative result would nearly exclude HIT. Another useful scenario for the PHA test is a patient with a high clinical probability of having HIT, but in whom the IgG specific (GHA) test is negative; in such a patient, a negative PHA also essentially excludes HIT. The IgG specific test (GHA) test offers high sensitivity (96%) and specificity (93%). An OD of 0.40 is defined as the positive threshold for the presence of anti-pf4/h antibodies in both the PHA and GHA tests. Currently the laboratory reports OD along with an interpretive result that reflects the relationship between antibody strength/titre and the likelihood that a positive test is clinically significant. As a general rule, a stronger OD increases the likelihood that the anti-pf4/h antibody detected is clinically significant (rising from <5% with an OD of 0.5 to ~95% with an OD of see Warkentin, et al). The specificity of a positive result is further enhanced by a confirmatory step in which excess heparin is added; if the reaction OD is reduced by >50%, heparin-specificity is confirmed. If the OD is reduced <50% by added heparin, the result is indeterminate. The clinical significance of an indeterminate heparin antibody test is not known, and may have multiple underlying causes, including non-pf4/h antibodies or weakly reactive heparin associated antibodies. Patients with a moderate to high probability of HIT whose serum is indeterminate for heparin antibodies and who require anticoagulation should receive a direct thrombin inhibitor agent. 5

6 Additional Caveats: (1) Patients receiving unfractionated heparin who do not develop thrombocytopenia should not be screened for the development of HIT antibodies due to the high frequency of seropositivity among all patients receiving UFH, most of which are due to non-pathogenic antibodies. (2) Blood smear examination may complement PHA/GHA testing in the initial evaluation of thrombocytopenic patients with possible HIT. For instance, the presence of dysplastic morphology or schistocytes suggests that an underlying primary bone marrow disorder or microangiopathic process, respectively, may represent alternative explanations for thrombocytopenia. (3) The serotonin release assay (SRA), a functional test for heparin-induced antibodies, is still considered the gold standard in HIT testing, but is usually not an option due to the 3-5 day turn around time for this send-out test - a potentially dangerous delay in a patient with clinical HIT if continued on UFH. In a clinically ambiguous situation, anti-coagulation with the direct thrombin inhibitor Argatroban can be instituted while waiting for the SRA. Monitoring of P2Y12 Inhibition (8816/P2Y) Therapy in Cardiac Patients Ref Range: P2Y12 Reaction Units (PRU): Patients on P2Y12 Inhibition Therapy: <231 Individuals not on P2Y12 inhibitor medications (mean + 2SD): The VerifyNow point-of-care device is available in the following Allina Health Metro Hospital laboratories for monitoring patients on anti-p2y12 therapy (clopidogrel, prasugrel, ticagrelor, ticlopidine): Core Lab at ANW, United and Mercy. The extent of ADP-induced platelet aggregation is recorded as the Platelet Reactivity Unit (PRU) value, which is diminished by thienopyridine agents that block the P2Y12 ADP receptor. It is important to note that the normal range among individuals not exposed to P2Y12 inhibitors is quite broad ( PRU). A recent meta-analysis found that patients on clopidogrel with PRU values <208 at 12 to 24 hours after PCI or during follow up have significantly fewer ischemic/thrombotic complications than those with values >208 (see Price et al). Failure to achieve PRU values <208 may reflect many factors causing resistance to P2Y12 inhibition, including genetic variation (cytochrome P 450 2C19 polymorphisms), certain medications (i.e., proton pump inhibitors, statins), other diseases (i.e., diabetes), and non-compliance. Previously, a % inhibition value was calculated using the patient PRU and the patient s base PRU (obtained by a non-p2y12 platelet agonist). In the spring of 2012 the device was modified to report only the patient PRU. Testing of P2Y12 inhibition should begin 5-7 days after beginning 75 mg/day of Clopidogrel or 2-6 hours after a 300 mg or 600 mg bolus. Up to 7 days may be required for complete restoration of platelet function following cessation of anti-p2y12 therapy. ADAMTS-13 and the Diagnosis of Thrombotic Thrombocytopenic Purpura (TTP) TTP is a microangiopathic thrombotic disorder resulting from either an inherited or acquired deficiency of the VWF-cleaving protease, ADAMTS-13. Acquired TTP most often occurs in the setting of an autoantibody against ADAMTS-13, whereas the inherited form reflects a mutation in either ADAMTS-13 itself or VWF. Several overlapping clinical syndromes share with TTP the finding of microangiopathic hemolytic anemia (MAHA) and thrombocytopenia, including the hemolytic-uremic syndrome (HUS), observed in children suffering from Shiga toxin-producing 6

7 E. coli, hypertensive disorders of pregnancy (HELLP), some drug-induced microangiopathies, malignant hypertension, and DIC. Timely diagnosis is crucial as a result of the high mortality of TTP (>90%) if left untreated versus the effectiveness of daily plasma exchange therapy. The urgency to begin therapy effectively restricts the diagnosis of TTP to a clinical one based on the classic pentad of MAHA, thrombocytopenia, neurologic symptoms, renal failure, and fever, supported by a few basic laboratory tests (see below). Surprisingly, determination of ADAMTS- 13 activity is of little practical value in the initial diagnosis of TTP, as some patients with clinical TTP have only modestly decreased ADAMTS-13 activity, while others with markedly diminished ADAMATS-13 levels may have no symptoms. Furthermore, the turnaround time of 3-5 days is too long to wait for the initiation of plasma exchange. In follow up, ADAMTS-13 determination may have prognostic value, however, as persistently low ADAMTS-13 activity following plasma exchange may predict for subsequent relapse. Caveat: In a blood smear with microangiopathy the differential diagnosis is frequently one of TTP (or one of its overlap syndromes) versus DIC. In this setting attention to the platelet count and basic coagulation results can be very helpful. While TTP typically presents with profound thrombocytopenia (<20,000/cu mm) and a normal or near-normal INR (<1.2), DIC more often correlates with moderate thrombocytopenia (>30,000/cumm) and a markedly abnormal INR (>2.0). Flow Cytometry Paroxysmal Nocturnal Hemoglobinuria (PNH) (2323/PNH) Ref Range: Negative for Immunophenotypic Evidence of PNH Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, clonal hematopoietic stem cell disorder with protean consequences that include bone marrow failure, hemolytic anemia, thrombocytopenia and thrombosis. Patients with untreated PNH are at high risk (up to 40%) for life-threatening venous thrombosis. Eculizumab, a humanized monoclonal antibody against the complement protein C5, is effective therapy for PNH. Flow cytometry has replaced the Ham s test in the evaluation of patients with suspected PNH. It is important to recognize that small populations of GPI-deficient (PNH type) blood cells can be found in the majority of patients with acquired aplastic anemia (usually % of granulocytes), up to 20% of patients with myelodysplastic syndromes (0.01-2% of granulocytes), and rarely in healthy controls (<0.01% of granulocytes). The Allina Health flow assay for PNH utilizes high-sensitivity multi-parameter flow cytometry for the semi-quantitative detection of two GPI-related proteins (CD55 and CD59) and an inactivated bacterial aerolysin probe (FLAER) that has high affinity for the GPI anchor. These GPI-related markers are queried on gated populations of granulocytes, monocytes, and red blood cells. Physicians may request flow cytometry only, with quantitative results, or with peripheral smear morphology correlation. Additional Flow Cytometric Studies of Platelet-Associated Antigens The flow lab at Allina Health Laboratory offers qualitative phenotype analysis of blood and bone marrow for patients with bleeding disorders who may have acute myeloid leukemia. In some cases platelet-associated markers (CD41, CD42, CD61) may be studied as part of a comprehensive leukemia evaluation including cytogenetics and morphology. Platelet flow cytometry can also be used to document loss or deficient expression of surface glycoproteins associated with Bernard Soulier syndrome (GPIb/IX/V) and Glanzmann 7

8 thrombasthenia (GPIIb/IIIa). These applications are currently not available at Allina Health Laboratory but can be obtained as part of a comprehensive evaluation including classical light transmission platelet aggregation via referral to the University of Minnesota. Von Willebrand Disease Von Willebrand factor mediates adhesion of platelets at sites of vascular injury and also stabilizes factor VIII (FVIII) in the circulation. Therefore, defects in VWF can cause bleeding by impairing platelet adhesion or by reducing FVIII levels. Not surprisingly, VWD may masquerade clinically as a platelet-associated bleeding disorder. Initial screening for VWD begins with the three-test Von Willebrand Screen (507/VWI) available at the Allina Health Laboratory, which includes measurement of the amount of VWF protein present in plasma (VWF:Ag), the function of the VWF protein as measured by ristocetin cofactor activity (VWF:RCo), and the ability of the VWF to serve as the carrier protein to maintain normal FVIII survival (factor VIII level). Routine coagulation testing (INR and APTT), platelet function testing (PFT), platelet count, and occasionally smear morphology are also appropriate initial tests in the workup of patients with bleeding disorders due to VWD. If either VWF:Ag or VWF:RCo are abnormal the specimen is automatically referred for multimer analysis at Mayo Medical Labs. A thorough review of the history, laboratory diagnosis, and clinical management of Von Willebrand Disease is available online: Platelet Aggregation Platelet aggregation measures the extent of platelet aggregation resulting from addition of a panel of agonists, and is useful in diagnosing primary platelet dysfunction syndromes resulting from abnormalities of surface glycoprotein expression, signal transduction, and platelet granule function. Currently, platelet aggregation studies are indicated as part of a more comprehensive evaluation performed at the University of Minnesota. 8

9 References Book: Hemostasis Testing An Algorithmic Approach Kandice Kottke-Marchant CAP Press, 2008 Journal articles: Kottke-Marchant, K and Corcoran, G. Archives of Pathology and Laboratory Medicine, volume 126 (2), Feb Heparin-Induced Thrombocytopenia: Warkentin TE, Greinacher A, et al. American College of Chest Physicians Evidence-Based Clinical Practice Guidelines (8th ed). Chest 2008;133 6 suppl:340s-380s. Lo, GK, JuhlL, D, Warkentin, et al Journal of Thrombosis and Haemostasis Vol 4, Issue 4. April Warkentin, TE ASH Education Book Dec 10, 2011 vol no P2Y12 Inhibition: Price, MJ, Angiolillo, DJ, et al Circulation 2011;124: Thrombotic Thrombocytopenic Purpura: Sadler, JM. Blood, 1 July 2008 Volume 112, No 1; For questions, comments, or suggestions about this newsletter or other laboratory issues, please contact Lauren Anthony, MD, Medical Director of Allina Health Laboratory, (612) or Lauren.Anthony@allina.com 9