From: Elizabeth M. Van Cott, M.D., and Michael Laposata, M.D., Ph.D., “Coagulation.” In: Jacobs DS et al, ed. The Laboratory Test Handbook, 5th Edition. Lexi-Comp, Cleveland, 2001; 327-358.
Related Information
Heparin-Induced Thrombocytopenia
Applies to Idiopathic Thrombocytopenic Purpura; Lymphocytotoxicity Assay; NAIT; Neonatal Alloimmune Thrombocytopenia; Platelet Transfusion Refractoriness; Post-transfusion Purpura; PTP
Abstract Platelet antibodies can be autoimmune (as found in idiopathic thrombocytopenic purpura (ITP)), drug-induced, or alloimmune (as found in neonatal alloimmune thrombocytopenia (NAIT), post-transfusion purpura (PTP), platelet transfusion refractoriness). Heparin-induced thrombocytopenia (HIT), the most common drug-induced immune thrombocytopenia, occurs by a unique mechanism. Therefore, HIT is discussed separately.
Specimen Varies depending on method. Whole blood for direct antibody tests (measuring antibody attached to platelets) or for identifying platelet antigens; serum or plasma for indirect antibody tests (measuring antiplatelet antibody not bound to platelets). DNA testing requires whole blood or other source of DNA.
Container Varies depending on method. Commonly requires lavender top (EDTA – whole blood or plasma) tube and/or red top (serum) tube. Some methods require 6-8 tubes of blood; other methods need only one tube. Specimens should be transported to the laboratory immediately.
Collection Routine venipuncture
Storage Instructions Varies depending on method. Some methods require whole blood at room temperature; others recommend refrigerated whole blood, or refrigerated or frozen serum or plasma.
Turnaround Time Usually several days, because these assays are often send-out tests.
Use Confirm drug-induced thrombocytopenia, NAIT, PTP, or platelet transfusion refractoriness. The tests are not considered necessary for diagnosing ITP, according to an expert panel.1 For ITP, the tests may lack adequate sensitivity (particularly certain newer methods) or specificity (particularly certain older methods).
Methodology A variety of methods exist. Older methods that measure antibody associated with platelets are generally sensitive but not specific.2 For example, the use of radiolabeled antibodies that bind to other antibodies are used in some specialized coagulation laboratories as either a direct or indirect antiplatelet antibody assay. More recently, a number of enzyme-linked immunosorbent (ELISA) assays are available to test for specific antiplatelet antibodies in serum or plasma. The platelet antigen of interest, such as an HLA antigen or glycoprotein Ib/IX, is bound to the surface of a microtiter plate. The patient sample is added and if antibody is present, it will bind to the antigen. In antigen capture immunoassays, monoclonal antibodies directed against platelet antigens are used to individually capture various known platelet antigens onto a solid phase. Patient serum is added. If the corresponding antibody is present in the patient serum, it will bind. For example, if an antibody in the patient serum binds in the assay containing the PlA1 antigen, then the patient is found to have an anti-PlA1 antibody. Flow cytometry is also used in some laboratories to detect platelet-associated antibodies.
NAIT: The diagnosis of NAIT often involves typing (identifying) platelet antigens in the mother and father (and newborn), to demonstrate that the mother lacks a platelet antigen that is present on the platelets of the father (and newborn). It can also be demonstrated that there is an antibody in the mother’s serum that is directed against a platelet antigen in the father (and newborn). Testing the newborn directly is typically not necessary, if the father can be tested.
PTP: The diagnosis of PTP often involves typing platelet antigens in the patient, and demonstrating that a platelet antibody in the patient’s serum is directed against an antigen that is absent on the patient’s platelets. Methods for detecting platelet antibodies in serum have been described above. Some of the current methods used for typing platelet antigens are described below.
Platelet antigen typing by antigen-capture immunoassays: Monoclonal antibodies are used to immobilize the patient’s platelet antigens onto a solid phase. Various antibodies of known antigen specificity are added. If an antibody binds, the patient’s platelets have that particular antigen. For example, if an anti-PlA1 antibody binds to the patient’s platelet antigens in this assay, then the patient is found to carry the PlA1 antigen. If the PlA1 antibody does not bind, then the patient’s platelets lack the PlA1 antigen. Alternatively, polymerase chain reaction (PCR) assays can be used to identify the patient’s platelet antigens. The platelet-specific antigens that cause platelet antibody formation are polymorphisms of platelet glycoproteins. Many of the alterations in DNA sequence that account for these polymorphisms are known and can be identified by PCR.
Drug-induced thrombocytopenia: The serotonin release assay, flow cytometry or other methods can be used to diagnose drug-induced thrombocytopenia. These tests are not routinely available. In serotonin release assays, patient plasma (or serum) and the suspected drug are added to normal platelets that contain radiolabeled serotonin. If antibodies against the drug are present, they stimulate the platelets to release their serotonin. The released radiolabeled serotonin can then be detected.
A lymphocytotoxicity assay (percent reactive antibody, PRA) can be used to detect HLA antibodies in patients who are refractory to platelet transfusions.
Additional Information
ITP: ITP is an isolated thrombocytopenia due to an autoantibody against platelets. The platelet antibodies are most commonly directed against components of platelet glycoprotein IIb/IIIa or to a lesser extent glycoprotein Ib/IX. In children, it is most often an acute disorder that resolves spontaneously. In adults, it is most often a chronic condition. Typically, the only abnormality on a peripheral blood smear is thrombocytopenia with normal to large platelets. Because ITP is a diagnosis of exclusion, laboratory tests recommended by a consensus panel to exclude other disorders include a peripheral blood smear, complete blood count (CBC), HIV testing in individuals with HIV risk factors, thyroid function tests in adults considering splenectomy, liver function tests in pregnant women to exclude HELLP syndrome, and bone marrow biopsy in persons older than age 60, adults considering splenectomy, or chronic cases in children that do not respond to IVIg.1
Neonatal alloimmune thrombocytopenia (NAIT) occurs when fetal platelets have an antigen from the father that is absent in the mother, and the mother forms antibodies that cross the placenta and destroy fetal platelets. Newborn platelet counts are often <100,000/microL at birth, returning to normal within 2 weeks after birth. The antigens are usually components of platelet glycoprotein IIb/IIIa, most commonly, an antigen called PlA1. The incidence of NAIT is approximately one case per 1000-5000 live births. See Platelet Transfusion.
Post-transfusion purpura (PTP) is a rare condition that occurs when a patient is transfused with platelets that express an antigen that is absent in the patient. The patient forms antibodies against the donor platelets. For unclear reasons in PTP, these antibodies also destroy the patient’s own platelets, even though they lack the offending antigen. As with NAIT, the antigens are usually components of platelet glycoprotein IIb/IIIa, most commonly PlA1. PTP is characterized by the sudden onset of thrombocytopenia 5-12 days after transfusion of a platelet-containing fraction. The thrombocytopenia is typically severe (<10,000/microL), and it usually begins to resolve within 14 days after the transfusion.
Drug-induced immune thrombocytopenia: A vast number of drugs have been implicated in drug-induced thrombocytopenia, but a cause-effect relationship has not been proven for most drugs. Some of the drugs that cause immune thrombocytopenia include quinidine, quinine, sulfonamides, sulfonylureas, gold salts, and salicylates. Some drugs cause thrombocytopenia through nonimmune mechanisms, including marrow suppression (eg, ethanol, thiazide, procarbazine) or nonimmune destruction (eg, ristocetin, bleomycin, protamine). In the nonimmune cases, there is no antibody and therefore no need for platelet antibody tests. With immune drug-induced thrombocytopenia, platelet counts are often severely decreased (<10,000/microL). Platelet counts typically return to normal within 7 days after discontinuing the drug.
Platelet refractoriness is a condition that occurs in thrombocytopenic patients who have received multiple platelet transfusions. The transfusions expose the patient to a variety of foreign HLA and other platelet antigens, against which the patient forms antibodies. These antibodies destroy subsequently transfused platelets, and, the patient is said to be refractory to platelet transfusion. Platelet refractoriness is most often due to antibodies against HLA-A or HLA-B antigens; less common causes include antibodies against ABO blood group antigens or platelet glycoproteins.
Footnotes
1. George JN, Woolf SH, Raskob GE, et al, “Idiopathic Thrombocytopenic Purpura: A Practice Guideline Developed by Explicit Methods for the American Society of Hematology,”Blood, 1996, 88(1):3-40.
2. Warner M and Kelton JG, “Laboratory Investigation of Immune Thrombocytopenia,”J Clin Pathol, 1997, 50(1):5-12.
References
Berchtold P, Muller D, Beardsley D, et al, “International Study to Compare Antigen-Specific Methods Used for the Measurement of Antiplatelet Autoantibodies,”Br J Haematol, 1997, 96(3):477-83.
Brighton TA, Evans S, Castaldi PA, et al, “Prospective Evaluation of the Clinical Usefulness of an Antigen-Specific Assay (MAIPA) in Idiopathic Thrombocytopenic Purpura and Other Immune Thrombocytopenias,”Blood, 1996, 88(1):194-201.
Bussel JB, Zabusky MR, Berkowitz RL, et al, “Fetal Alloimmune Thrombocytopenia,”N Engl J Med, 1997, 337(1):22-6.
Moore SB and DeGoey SR, “Serum Platelet Antibody Testing. Evaluation of Solid-Phase Enzyme Immunoassay and Comparison With Indirect Immunofluorescence,”Am J Clin Pathol, 1998, 109(2):190-5.
Taaning E and Svejgaard A, “Post-transfusion Purpura: A Survey of 12 Danish Cases With Special Reference to Immunoglobulin G Subclasses of the Platelet Antibodies,”Transfus Med, 1994, 4(1):1-8
