Impact-R determines the degree of platelet adhesion and platelet aggregation under near physiologic conditions. Platelet adhesion is expressed as %SC or the percentage of the surface covered by platelet aggregates. Platelet aggregation is expressed as AS µm² or the average size of the aggregates.

Ristocetin.

Sometimes, a platelet agonist is needed to assay platelet function in cases where there is defective adhesion and aggregation. One of these agonists is Ristocetin. Ristocetin is an antibiotic previously used to treat staphylococcal infections. Use was discontinued because of its lethal side-effects, thrombocytopenia and platelet agglutination. Because of these same side-effects, ristocetin is now used to diagnose certain hematologic conditions such as von Willebrand disease and Bernard-Soulier syndrome.

Ristocetin causes von Willebrand factor to bind the platelet receptor GpIb so that when ristocetin is added to whole blood, it agglutinates. The mechanism for such activity is yet unexplained but there are several hypotheses including one in which the binding of ristocetin to the platelet changes its surface charge. Ristocetin will show hypoagglutination in von Willebrand disease because of a deficiency in von Willebrand factor and in Bernard-Soulier syndrome because of a deficiency in GpIb receptor proteins in the platelet cell membrane.

Tests that use ristocetin include: Ristocetin Cofactor Activity and Ristocetin Induced Platelet Aggregation.

Thrombin is a powerful platelet agonist.

Impact R is a useful tool to assess platelet function in different hematologic diseases and to monitor anti-platelet therapy.  In the study by Shenkman, et.al., Impact-R was used to test agonist-induced platelet aggregation.

Some of the known platelet agonists are: ADP, epinephrine, arachidonic acid, gamma-thrombin, and collagen. In the aforementioned study, they used ADP, ristocetin, epinephrine, collagen and arachidonic acid. These agonist act through various mechanisms that affect different stages of the coagulation process.

The Impact-R agonist response test detected platelet aggregation defects in patients with storage pool disease, von Willebrand disease and epinephrine response deficiency.  It may be useful in determining response to various platelet agonists.

Platelet apheresis concentrates are an invaluable blood product in the treatment of many disorders involving platelet deficiency. Congenital diseases such as Glanzmann’s thrombasthenia, Platelet type von Willebrand disease, and May-Heggelin anomaly, as well as acquired diseases such as Dengue Hemorrhagic Fever rely on platelet transfusions.

A previous article focused on the testing of platelet concentrates as they are stored in the laboratory facility. A study by Horvath, et.al., however, tested the patient’s blood for platelet function after a platelet transfusion has been done. In many cases, the effect of a platelet transfusion is quantified as an increase in the platelet count and not the platelet function.

The use of Impact-R as a diagnostic and monitoring tool for platelet function is feasible as was concluded by Horvath, et.al.

Statins are a class of drugs that lower blood cholesterol by inhibiting the enzyme HMG-CoA reductase. This enzyme, found in the liver, plays an important role in the production of cholesterol. It reduces HMG-CoA to mevalonate, and this conversion is the rate-limiting step in cholesterol biosynthesis.

HMG-CoA reductase pathway and drugs that inhibit the various steps (in red).

Statins act by competitive inhibition of HMG-CoA reductase since the statins are molecularly similar to HMG-CoA. Statins are used to lower cholesterol levels in patients who have tried diet and lifestyle modifications but still have high levels of cholesterol.

Medical use of statins is to decrease mortality in patients with preexisting cardiovascular disease and those who are high-risk for developing heart disease. Researchers propose four mechanisms for reduced cardiovascular events in patients taking statins:

1. Improve endothelial function
2. Modulate inflammatory responses
3. Maintain plaque stability (in atheromas)
4. Prevent thrombus formation

It is the 4th mechanism that is the subject of a study by Matetzky, et.al., which examined the effects of statins on platelet adhesion and aggregation. This study utilized the IMPACT-R to determine aggregate size and surface coverage of platelets taken from hypercholesterolemic and STEMI (ST-elevation myocardial infarction) patients treated with statins as compared to patients not treated with statins. An abstract of the study can be found here.

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.

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.