The study of platelet alterations has benefited from consider able progress in recent decades, mainly due to important acquisitions in physiopathology, new knowledge in molecular biology, and the development of innovative technologies applied to instruments that have progressively spread in lab oratories. The starting point, however, is the complete CBC with microscopic observation of the peripheral blood smear. From these initial tests, it is possible to obtain information on the diagnosis of two main platelet diseases: immune thrombocytopenic purpura, also known as Werlhof’s disease, and thrombotic thrombocytopenic purpura (TTP). In addition, immunoglobulin measurements are sometimes required in pediatric patients to rule out common variable immunodeficiency as a cause of thrombotic thrombocytopenic purpura. Careful observation of the peripheral smear is essential in a patient with thrombocytopenia. It can rule out forms of pseudothrombocytopenia, such as those due to platelet satellites, in which platelets adhere to neutrophils, causing a false reduction in count on CBC (Fig. 1). Moreover, giant platelets could be found in patients with immune thrombocytopenic purpura; in this case, they are not specific markers of the condition but rather a sign of the stimulus exerted by thrombopoietin on the bone marrow due to thrombocytopenia (Fig. 2).
Fig1. Microscopic image of a blood smear from a patient with pseudothrombocytopenia from satellite
Fig2. Giant platelet in peripheral smear. Normal-sized platelets on the right are observed for comparison
In thrombotic thrombocytopenic purpura, fragmentation of red blood cells (schistocytes) due to peripheral microangiopathy, as well as thrombocytopenia, could be observed. The minimum criteria for the diagnosis of thrombotic thrombocytopenic purpura are thrombocytopenia and microangiopathic hemolytic anemia without apparent etiology. In the complete form, neurological abnormalities, high fever, and acute renal failure occur. A smear examination shows thrombocytopenia and the presence of schistocytes. In addition, increased LDH and reticulocytosis are observed. Signs of intravascular coagulation are absent or only slightly increased in patients with TTP.
Rare congenital thrombocytopenia, known as Bernard Soulier syndrome, which causes easy nose and gum bleeding as well as gastrointestinal bleeding, can also be suspected based on the examination of the peripheral smear (Fig. 3) when thrombocytopenia is accompanied by the presence of giant platelets. This syndrome is due to a molecular defect in which the glycoprotein complex of the platelet membrane, gp Ib-gp V-gp IX is missing, with a consequent inability to bind to von Willebrand factor (VWF) and therefore to adhere to the platelet. The genes involved are GP1BA, GP1BB, and GP9, which encode for gp Ibα, gp Ibβ, and gp IX subunits. The bleeding time is prolonged. A careful smear examination is essential to exclude thrombotic thrombocytopenic purpura and rare cases of acute leukemia that initially present with thrombocytopenia.
Fig3. Giant platelet in a patient with Bernard–Soulier syndrome
Antiplatelet antibodies responsible for auto-immune thrombocytopenia (ITP) do not induce complement- mediated lysis. Patients with autoimmune thrombocytopenia have larger platelets than normal ones, with increased mem brane surface area and an increased number of Fc receptors than normal platelets. For these reasons, any increase in platelet-related immunoglobulins (antiplatelet antibodies) is not useful for diagnostic purposes because they are increased in almost all conditions associated with thrombocytopenia. This limits the value of the antiplatelet antibody test in the diagnosis of immune thrombocytopenic purpura.
One form of acquired thrombocytopenia that often comes first to the attention of the laboratory is heparin-induced thrombocytopenia. This is a particular alteration usually observed in samples from surgical (but also medical) patients who have received some form of heparin for prophylactic/ therapeutic purposes, although the highest frequency is observed with unfractionated heparin and can affect up to 5% of patients. The cause is due to the exposure of a particular masked epitope that occurs in some individuals because of the binding that normally takes place between heparin and PF4. The generation of antibodies, in response to this epitope exposure, proceeds with the formation of circulating immune complexes that activate platelets through binding to the Fc receptor. Platelet activation continues with further release of PF4, and the phenomenon tends to amplify, causing thrombocytopenia due to the aggregation of additional platelets and hypercoagulability that can result in thrombosis. Heparin-induced thrombocytopenia appears approximately 5–10 days after the initiation of drug administration. A rapid- onset form of heparin thrombocytopenia is anamnestically observed in patients who had previously received heparin without developing thrombocytopenia. The immediate measure is to discontinue heparin administration. The risk is much lower with fractionated low molecular weight heparins. The risk is absent when using one of the direct anti-FXa anticoagulants (e.g., rivaroxaban) for the same indications.
A rare form of spurious thrombocytopenia that usually comes first to the attention of the laboratory is that resulting from samples collected in EDTA. This phenomenon is due to the presence of EDTA-dependent antiplatelet antibodies that are activated at low temperatures. They cause agglutination of platelets and, therefore, a lowering of count values in automated instruments. It is suspected when unexplained thrombocytopenia, without clinical signs (absence of petechiae, normal bleeding time), is observed in outpatients. In order to ascertain whether EDTA is the cause of thrombocytopenia, it is necessary to take a second sample with a different anticoagulant (e.g., sodium citrate) and perform the count again, which will give a normal value if positive.
Bleeding time is the most widely used test for assessing primary hemostasis. However, despite its theoretical useful ness, this test is highly operator-dependent and not recommended as a screening test in the diagnostic routine for disorders of hemostasis. Bleeding time is measured by measuring at 30-s intervals the time required to observe the cessation of bleeding following a 1-mm-deep, 1-cm-long superficial cut on the volar surface of the forearm made by a standard cutter. Under these conditions, cessation of bleeding results from the formation of a primary hemostatic plug. There is a fairly linear correlation between bleeding time and platelet count between 10,000/μL and 100,000/μL. Bleeding time is prolonged with platelet counts below 75,000/μL, although this does not provide an explanation as to why the platelet count is low. In patients with thrombocytopenia, bleeding time should not be performed as it has no diagnostic significance. In alterations of secondary hemostasis related to coagulation mechanisms (e.g., in patients with hemophilia A or B), the bleeding time is always normal.
