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.

Platelets are small, granulated bodies of two to four micro meters in diameter, present in the blood along with other cellular components. About 300,000 per micro liters platelets are present in the circulating blood. Their normal half life is about four days.

Platelet production is being carried out in the bone marrow, where the megakaryocytes, giant cells form platelets by pinching off bits of cytoplasm and extruding them into the circulation. Certain colony stimulating factors regulate the platelet production. Out of these, a newly cloned factor is the thrombopoeitin, which facilitates megakarycyte maturation.

Between 65 to 70% of the platelets extruded from the bone marrow circulates in the blood, but the remainders are mostly in the spleen. So, splenectomy causes increase in the circulating blood platelet count (thrombocytosis).

At the event of blood vessel wall injury platelets adhere to the exposed collagen, laminin and von Willebrand factor in the wall via integrins on the platelet surface. Unlike platelet aggregation, the process of platelet adhesion is not an active process. It does not require any platelet metabolic activity. However, when the platelets bind to collagen, it initiates platelet activation, necessary for platelet aggregation. Activation of the platelets can also be produced by ADP and thrombin. By changing their shape, putting out pseudopodia and discharging granules, the platelets stick to other platelets. This process is known as platelet aggregation. It is suggested that the process of aggregation can also be fostered by the platelet activating factor (PAF), a cytokine secreted by neutrophils, monocytes and platelets themselves.

Decreased platelets result in deficient clot retraction and poor constriction of ruptured vessels. It results in the clinical syndrome commonly called as thrombocytopenic purpura, which is characterized by easy bruisability and multiple subcutaneous hemorrhages. Purpura may occur in the cases where the count is normal but the circulating platelets are abnormal (thrombasthenic purpura).

Here I would like to discuss a remarkable product Impact-R which is widely used for the assessment quantity and functionality of the blood platelets. Abnormal functioning platelets are detected with the help of Impact-R, which would otherwise lead to serious hemostatic abnormalities.

Blood cells are produced in the bone marrow. The three main components of blood are the red blood cells which carry oxygen, white blood cells or leukocytes which fight infection and platelets also called thrombocytes which assist in the formation of blood clot.

When a blood vessel is damaged platelets adhere to the surface of damaged vessel wall and release chemicals. The chemicals attract more platelets as well as red blood cells in order to form a clot or thrombus. As the clot grows the blood vessel narrows, thereby decreasing the blood loss. This process is called coagulation.

Normal platelet counts are in the range of 150, 000 to 350, 000 platelets per micro liter. Thrombocytopenia or low blood platelets are the disorders in which there are not enough platelets in the blood. When the platelet count is decreased the body is unable to form blood clots and is therefore unable to control the bleeding. Bruising and bleeding can occur from relatively little trauma. When the platelets count gets below 10, 000 platelets per micro liter, bleeding can develop even without significant trauma.

Chemotherapy induced thrombocytopenia is a disorder that develops as an adverse effect of chemotherapy. Cancer drugs not only kill cancer cells, they can also damage the platelet forming cells in the bone marrow. The severity of this disorder depends on the type of chemotherapy and the duration of treatment. Fortunately chemotherapy induced thrombocytopenia or low blood platelets can be managed with platelet transfusions, additional medications such as blood cell growth factors or with blood stem cell transplants.

Other common causes of thrombocytopenia or low blood platelets are ITP (immune thrombocytopenic purpura) and heparin induced thrombocytopenia. In immune thrombocytopenic purpura, anti platelet antibodies coat the platelets and destroy them, while heparin induced thrombocytopenia is caused by the formation of abnormal antibodies that activates platelets.

Impact-R has evolved as a blessing in disguise for the thrombocytopenia hit populations as it provides a very effective screening test for the timely detection of the patients suffering from thrombocytopenia. It has markedly decreased the morbidity and mortality associated with this disorder.

Al l four serotypes of dengue viruses are well known for taking the toll of healthcare facilities all over the world. Sporadic cases of dengue fever have scattered distribution including US, Mexico, Puerto Rico and you name any place. Mankind has also seen a multitude of dengue fever epidemics especially in 1979 and 1980 when they swept Asia, Africa and North America. Now, once again dengue has hit a gorgeous land of Pakistan, especially the Punjab Province.

As already mentioned dengue virus has four serotypes (dengue 1-4), having Aedes aegypti as their principal vector. Aedes aegypti is commonly known as the ‘Tiger mosquito’ that bites during the day as compared to the Anopheles species (causing malaria) that usually bite at night. Aedes aegypti is also a vector for the Yellow fever virus and the Chikungunya virus. It typically breeds near human habitation, using relatively fresh water from the sources like vases, jars, containers etc. All the four serotypes cause the same type of disease in the human beings.

The classic dengue fever has an incubation period of 2-7 days, and patient experiences sudden onset of fever, headache, retro orbital pain, severe back pain associated with myalgias. Due to these symptoms, the fever has been given the name of ‘Break Bone fever’. Other associated features of this disease may be anorexia, nausea, vomiting, and appearance of maculopapular rash all over the body, scleral injection and adenopathy. Even epistaxis and petechiae may appear in an uncomplicated disease. Laboratory findings are leucopenia, thrombocytopenia and in some cases serum aminotransferases may be elevated.

Thrombocytopenia is an important diagnostic finding in the cases of dengue fever that occurs due;

• Direct infection of bone marrow megakaryocytes.
• Immunological shortened platelet survival.
• A more complicated form of infection is the Dengue Hemorrhagic Fever/Dengue Shock Syndrome. It can occur in two forms;
• Re infection of an individual by a heterologous strain of the dengue virus previously infected from the dengue virus.
• Even classic dengue fever can progress to dengue hemorrhagic fever.

Dengue hemorrhagic fever is characterized by overt bleeding in the absence of underlying causes, increased bleeding tendency (can be appreciated by tourniquet test or presence of petechiae). In mild cases restlessness, lethargy, thrombocytopenia (platelets < 100,000 per micro liters) and hemoconcentration appears usually 2-5 days after classic dengue fever. In severe cases there is frank shock, low pulse pressure, cyanosis, hepatomegaly, pleural effusion and ascites. Individuals usually bleed profusely in some severe cases. Most of the people usually respond well on supportive therapy, however some need platelet transfusions.

Here comes the role of Impact-R which can be used for clinical diagnosis of dengue fever by confirming the presence of thrombocytopenia in an individual. Moreover, it also helps determining the function of the platelets being infused in the patients. Stored platelets are usually used in the patients for the treatment of dengue hemorrhagic fever and their functional assessment is done by Impact-R.

Patients with end stage renal disease suffer from a multitude of haemostatic disorders. This bleeding is mainly due to impaired platelet function present in the uremic patients. Both the platelets adhesion (platelet vessel wall interaction) and the aggregation are affected resulting in bleeding diathesis. Studies have shown that haemodialysis of these patients in time results in the decreased hemorrhagic episodes.

Impact-R is can be utilized in these cases for the early detection of platelet function abnormalities, and can help in reducing the morbidity and mortality associated with hemorrhage in end stage renal disease.

Before discussing the causes of low blood platelets or thrombocytopenia, I feel necessary to mention the grave consequences associated with low blood platelets. The dominant features of thrombocytopenia include petechial cutaneous bleeding, intracranial bleeding and oozing from mucosal surfaces.

Causes can be divided into three main groups;

• Decreased production
• Increased destruction
• Un replaced loss or dilution of platelets

The characteristic findings of thrombocytopenia are decreased platelet count and prolonged bleeding time. Bone marrow aspiration is helpful in the cases of thrombocytopenia. It reveals decreased megakaryocytes (immature form of platelets) when caused by decreased platelet production and increased megakaryocytes when caused by increased platelet destruction.

Now I would like to discuss few common causes of low blood platelets;

• Irradiation, exposure to drugs or chemical causes decreased production of the platelets.
• Acute leukemia in which there is decreased platelet production due to replacement of bone marrow by blast cells.
• Myelophthisis results in low blood platelets because the bone marrow is replaced by tumor cells.
• Aplastic anemia can also be the cause of low blood platelets. It may be caused by exposure to toxic agents like benzene or due to autoimmune destruction by cytotoxic T cells.
• Circulating blood platelets are lost or destroyed when they are sequestered by spleen, thus decreasing the blood platelet count.
• Dilution of platelets occurs in multiple transfusions, where there is relatively deficiency of platelets and thus low blood platelets.
• Disseminated intravascular coagulation, commonly known as consumptive coagulopathy results in consumption of almost all the platelets in the blood.
• Low blood platelets can be secondary to other disease such as acquired immunodeficiency syndrome or systemic lupus erythematosus.
• Immune thrombocytopenic purpura is a very common autoimmune disorder of the platelets occurring due to formation of anti platelet antibodies in the blood, which damage the platelets and decreases their count.

All the above mentioned causes of thrombocytopenia can be diagnosed well before time by the use of Impact-R platelet analyzer. This kit uses the very basic principles of platelet aggregation and adhesion and can be used to screen the cases of thrombocytopenia due to any cause.