One cannot discount the importance of platelet concentrates as a treatment for many bleeding disorders. Many patients with platelet dysfunctions rely on platelet transfusions. A case in point, platelet concentrates are a lifesaving measure in patients with Dengue Hemorrhagic Fever.

Platelet concentrates are ideally transfused as soon as they are collected but can be stored for up to 5 days when kept on a rotator at 20-24 degrees Celsius. It is important that platelets be kept in continuous gentle rotation to keep them suspended in plasma and prevent aggregation.

Issues with transporting and storing platelets may be a stumbling block in areas where a pheresis machine is not readily available. Platelet concentrates are expensive to obtain and its use should be optimized. One way to make sure that the platelet concentrates are still potent is through testing and monitoring its adhesion and aggregation capabilities. Studies have been done using the IMPACT-R machine as a monitoring tool. An abstract of one such study can be found here.

Dohle body in the cytoplasm of a granulocyte.

The Inherited Macro-thrombocytopenias are autosomal dominant disorders characterized by mild to moderate thrombocytopenia with large platelets and varying degrees of platelet dysfunction, usually an absence or reduction of the secondary wave of platelet aggregation resulting in little or no bleeding. Thrombopenic symptoms are purpura, ecchymoses, epistaxis and heavy menstrual bleeding. Some may also have leukocyte inclusions, interstitial nephritis, sensorineural hearing loss and cataracts.  Mutations in the heavy chain IIA MYH9, a prominent non-muscle myosin, result in a spectrum of disorders with variable manifestations.

May-Heggelin anomaly (MHA) is characterized by macrothrombocytopenia and presence of Döhle bodies in the cytoplasm of leukocytes. Most of the time, MHA requires no treatment but in extreme cases, a platelet transfusion may be beneficial.

Fechtner syndrome includes a macrothrombocytopenia with smaller calibre platelets than MHA, leukocyte inclusions, interstitial glomerulonephritis, sensorineural deafness, and cataracts. Treatment includes specific management of the components (e.g. lens replacement for cataract) and platelet transfusions if contemplating surgery.

Sebastian syndrome is purely hematologic in manifestation. It involves macrothrombocytopenia which is usually asymptomatic and leukocyte inclusion bodies that are smaller than those seen in MHA, may be numerous and are generally difficult to stain.

Epstein syndrome has nephritis, hearing loss, platelet adhesion and aggregation defects but no leukocyte inclusions are demonstrated. Eckstein syndrome has nephritis and deafness but no platelet dysfunction. Enyeart syndrome has thrombocytopenia and giant platelets with inclusions but no leukocyte inclusions.

Cytoplasmic inclusions are formed when the unstable abnormally dimerized MYHIIA protein precipitates with the normal MYHIIA.

Giant platelet.

Abnormal dimerization also brings about failure to properly organize the cytoskeleton of megakaryocytes, which causes giant platelets and thrombocytopenia.

Since these disorders are autosomal dominant with phenotypic consequences that are not readily apparent, screening tests for siblings or offspring of known affected individuals may be an important step. In this regard, Impact-R can be of much use. It is a cone and platelet analyzer that qualitatively determines platelet adhesion and aggregation. A peripheral smear alone may not predict the need for platelet transfusions in patients about to undergo surgery. Impact-R could also be of much use in further research for these conditions especially in answering the question, “How is adhesion and aggregation affected by abnormal platelet size?”

Inherited platelet disorders are rare conditions that are not usually encountered in clinical practice. However, the study of the pathophysiology has led to a better understanding of platelet biochemistry and physiology. They may be subdivided into the following:

1. Platelet Membrane Disorders
2. Platelet Granule Disorders
3. Macrothrombocytopenias
4. Platelet Signaling Disorders

The Platelet Membrane Disorders:

Glanzmann’s Thrombasthenia is a rare disorder where platelets can carry out biochemical reactions but are unable to form aggregates. This is an autosomal recessive trait where platelets have absent or dysfunctional GpIIb/IIIa complexes.

Smooth inactivated platelet (blue) with spiky activated platelets (light blue). ©2000 Dennis Kunkel, Ph.D.

In a normal platelet, there are about 50,000 of these complexes in the membrane. When platelets are activated, the complex binds fibrinogen, which in turn bind to GpIIb/IIIa complexes on other platelets, resulting in multicellular aggregates. Platelets are normal in size, shape and number. Patients with Glanzmann’s Thrombasthenia have mucosal bleeding throughout life and may even have severe bleeding episodes requiring platelet transfusions.

Bernard-Soulier Syndrome is another rare autosomal recessive disorder caused by mutations of the GpIb/IX/V complex. This complex is the main receptor for von Willebrand’s factor, which anchors platelets to exposed subendothelium in cases of endothelial injury and under high shear stress. The platelets are abnormally large and the count is low. Patients present with muco-cutaneous bleeding and prolonged bleeding time. They may require platelet transfusions and sometimes respond favourably to Desmopressin.

Pseudo- or Platelet type von Willebrand Disease is an autosomal dominant disorder arising from mutations of the GpIba polypeptide that make the platelet hypersensitive to vWF. Mucosal bleeding and borderline thrombocytopenia are noted. Pseudo-vWD resembles Type IIB vWD. An accurate diagnosis is necessary since treatment differs, Pseudo-vWD requiring platelet transfusions while Type IIB vWD requires vWF transfusion.

ADP receptor Deficiency is an autosomal recessive disorder involving the ADP receptor P₂Y₁₂. ADP released from damaged tissues and activated platelets plays a role in platelet aggregation through the mediation of receptors P₂Y₁ and P₂Y₁₂.   P₂Y₁ initiates platelet response to ADP while P₂Y₁₂ forms and sustains large aggregates. Patients, therefore, have mild bleeding but are susceptible to posttraumatic and post-surgical blood loss.

Collagen receptor deficiency involves two receptors, an integrin protein, GpIa/IIa, and a non-integrin protein, GpVI. This deficiency is still under intense study.

Platelet function studies that show how platelets behave in vitro can be very helpful in diagnosis of these conditions. One of these is studying the platelet aggregates formed when blood is exposed to polystyrene in flow conditions. The IMPACT-R machine is one laboratory equipment that uses the cone and plate principal.

Impact-R may be used in platelet function studies of patients with Storage Pool Disease.

Platelet Storage Pool Disease(SPD) involves disorders with reduction in the number and contents of platelet granules. These may be a-granules, d-granules, or a combination of both. The most common of which is d-SPD, and  the rarest is ad-SPD. The Gray Platelet Syndrome is an a-SPD where there is severe reduction of the number and contents of a-granules. The defect can be

Hermansky-Pudlak syndrome.

of two modalities, one wherein the SPD is restricted to the platelets and another where it is part of systemic syndromes with defective assembly and packaging of granules. An example would be Hermansky-Pudlak syndrome where the patient has oculocutaneous albinism, bleeding disorders, and cellular storage disorders.

SPD is an autosomal recessive trait but can also be an acquired disorder.  It may be seen in patients with myeloproliferative disorders, myelodysplasia, acute leukemia, and even systemic lupus erythematosus (SLE).  Treatment is supportive, and may involve platelet transfusions.

IMPACT-R is a cone and plate analyzer that can be used for the study of platelet function, either hyper- or hypo-function. It is an important screening tool for platelet abnormalities and may be used as a monitoring tool for therapeutic response in patients being treated.

A novel method to test for platelet function may be an important adjunct in the diagnosis of and research about ITP. This is the CPA or Cone and Plate Analyzer (IMPACT-R) which tests for platelet adhesion and aggregation under simulated arterial flow conditions.

Immune Thrombocytopenic Purpura (ITP) otherwise known as Idiopathic Thrombocytopenic Purpura, is an acquired platelet disorder. This involves immune-mediated destruction of platelets as well as inhibition of platelet release from the megakaryocyte. The exact nature of the immune dysfunction is not known but is usually associated with infections and auto-immune disorders. Common infections causing ITP are Human Immunodeficiency Virus and Hepatitis C.  Systemic Lupus Erythematosus is the most common auto-immune disorder implicated.

ITP usually presents as an acute disease in children and runs a more chronic course in adults. Mucocutaneous bleeding in the gastrointestinal tract (gum bleeding, oral mucosal bleeding, etc.), and in the female reproductive tract (heavy menstrual bleeding) are seen. Patients also note ecchymoses and petechiae. Thrombocytopenia on a routine CBC may be the only sign

Blood blister under the tongue

prompting a consultation. Life-threatening bleeding very rarely occurs and is usually heralded by oral blood blisters and retinal hemorrhage.

A careful and comprehensive history and physical examination will reveal a high index of suspicion for ITP. Laboratory testing to confirm the diagnosis includes: complete blood count with a peripheral blood smear, bone marrow biopsy, clotting time and bleeding time. The peripheral smear will show large platelets with normal morphology. Laboratory tests to look for the secondary causes of ITP are recommended. These are: testing for HIV and Hepatitis C, serologic testing for SLE.  If anemia is present, a Coomb’s test is done to rule out Evan’s Syndrome. This syndrome is a combination of ITP and autoimmune hemolytic anemia.

The new Impact-R cone and plate analyzer can be of use in screening for ITP and also for monitoring therapeutic response.

Treatment for ITP depends on severity. Mild ITP, or those without significant bleeding symptoms and severe thrombocytopenia, can be treated in an outpatient setting. Agents include Prednisone, Rh₀(D) immune globulin, intravenous gammaglobulin (IVIgG). Severe ITP requires hospitalization and treatment with high-dose glucocorticoids, anti-Rh₀(D), IVIgG, and immunosuppressive agents such Rituximab. Patients who relapse after medical therapy may benefit from a splenectomy.

Impact-R CPA test can be a useful screening test for von Willebrand Disease.

Von Willebrand Disease or vWD is the most common hereditary bleeding disorder. It is caused by a deficiency in the von Willebrand Factor (vWF).  VWF has two main functions in the coagulation cascade: 1) it is the major adhesion molecule that anchors the platelets to the exposed subendothelium, and 2) it is the binding protein for Factor VIII (FVIII). The clinical manifestations of vWD is due to its effect on platelet function.

Patients with vWD present mostly with mucosal bleeding and sometimes with post-operative bleeding.  Signs and symptoms include: bruising in uncommon areas of the body, prolonged epistaxis, heavy menstrual bleeding or menorrhagia, prolonged bleeding at dental extraction sites, or during tonsillectomies.

VWD has 3 major types (Type 1, 2, and 3) with Type 2 having 4 subtypes (Type 2A, 2B, 2M, and 2N). The most common is Type 1. The following table compares the Activated Partial Thromboplastin Time (aPTT), vWF antigen, vWF activity, and FVIII activity of the different types.

Von Willebrand factor replacement is the definitive therapy for vWD especially as prophylaxis for major procedures to reduce the risk of post-operative bleeding. For minor procedures and for mild Type 1 vWD treatment of choice is 1-deamino-8-D-arginine vasopressin (DDAVP or desmopressin) which causes the release of vWF and FVIII from endothelial stores. It can be given intravenously or by an intranasal spray.

Diagnosis is done by careful history and physical exam with a combination of several laboratory tests such as aPTT, ristocetin cofactor assay, collagen binding activity multimer assays. A useful screening test to determine decreased platelet function is the IMPACT-R Cone and Platelet Analyzer (CPA) which tests platelet adhesion and aggregation in anti-coagulated whole blood under arterial flow conditions. This test is qualitative compared to the merely quantitative platelet count.