What is a screening test? It is a device used to detect disease or a precursor of a disease in persons who do not have signs and symptoms. The decision to screen should be based on a careful history and assessment of risk factors.

Children with a family history of bleeding disorders may well benefit from a

Alexei Nikolaevich, heir to the Russian throne, had Hemophilia B.

screening test. The child’s future health and quality of life will depend on the diagnosis of a serious medical condition ahead of the time when signs and symptoms manifest. Children with a family history of congenital platelet function defects, clotting factor deficiencies, and defects in primary or secondary aggregation are among those that will put a screening test to good use.

A study by Revel-Vilk, et.al. evaluated the use of the Impact-R as a one such screening test. They concluded that the Impact-R is highly effective in excluding a bleeding disorder in patients with normal result. However, in case of abnormal Impact-R results, further testing is necessary to diagnose the condition.

The aortic valve is the valve between the left ventricle and the aorta, the main artery. It has three leaflets (tricuspid). When the left ventricle contracts, it pushes blood through the valves and into the aorta. When the left ventricle relaxes, the valves close, preventing the blood from flowing back into the left ventricle.

When the aortic opening becomes constricted or stenotic, the blood in the left ventricle cannot be pumped effectively into the aorta. Aortic stenosis (AS) is usually age-related and is caused by progressive calcification of any of the three leaflets of the valve.

Severe aortic stenosis also causes an acquired form of von Willebrand disease (vWD type 2A). This is due to breakdown of the von Willebrand factor (vWF) by the increased turbulence around the stenotic valve. The increased turbulence causing high shear stress conditions predisposes the vWF to cleavage by an enzyme, ADAMTS-13. This enzyme is the 13^th member of the class of enzymes known as a disintegrin and metalloproteinase with a thrombospondin type 1 motif also known as von Willebrand factor-cleaving protease (vWFCP).

This acquired form of vWD causes bleeding in the AS patient. Whether the bleeding is due to platelet adhesion or to platelet aggregation has not been determined. This is the subject of the study done by Panzer, et.al. using the IMPACT-R machine. The study concludes that reduced levels of large vWF multimers associated with severe AS leads to impairment of both adhesion and aggregation of platelets. An abstract of the study can be found here.

Philippe Gaucher, A French doctor first described a condition in a 32-year old woman with an enlarged spleen in 1882. This condition would soon be known as Gaucher’s disease and its biochemical nature elucidated in 1965.

Gaucher’s disease (GD) is an autosomal recessive disorder in chromosome 1

Acid beta-glucosidase.

affecting the enzyme acid beta-glucosidase (a.k.a. lysosomal glucocerebrosidase, glucosylceramidase, D-glucosyl-N-acylsphingosine glucohydrolase). This enzyme catalyses the breakdown of glucosylceramide, a component of the cell membranes of WBC and RBC. When a macrophage phagocytoses the WBC and RBC that are due for destruction, glucosylceramide accumulates in the macrophage and cannot be eliminated.  Glucosylceramide can also accumulate in the brain  due to the rapid turnover of complex lipids during brain development and myelin sheath formation.

There are three types of GD, types I, II and III, of which type I is the most common and the least debilitating form. Unlike the other two, type I is non-neuropathic. Symptoms and signs are noted early in life or in early adulthood and includes hepatosplenomegaly, frequent infections, anemia, leukopenia and thrombocytopenia, osteoporosis and arthralgias, yellowish-brown skin pigmentation, and frequent ecchymoses and mucosal bleeding. The bleeding symptoms are sometimes noted to be disproportionate to the level of thrombocytopenia.

A research done by Spectre, et.al. proposes to clarify why the degree of mucosal bleeding does not coincide with the platelet count. His team noted that it is a defect in platelet adhesion that may be a potential cause for the bleeding. The study included patients with type I GD (with a platelet count of more than or equal to 130,000 per liter and a hematocrit of more than or equal to 30%) and healthy controls. They noted that type I GD patients had significantly lower platelet adhesion (as determined by IMPACT-R) that was not improved even with enzyme replacement therapy but was improved after splenectomy. Many of these patients also had mucosal bleeding. An abstract of the study can be found here.

What is FMD?

Brachial artery scanning to measure FMD.

Flow-Mediated Dilatation is the increase in calibre of a blood vessel in response to increased blood flow. This was first described by Schretzenmayr. FMD is endothelium dependent, wherein the endothelium acts as a mechanotransducer that senses changes in shear stress and then determines the release of dilatators. Several dilator factors have been proposed to be involved in FMD: prostaglandins , ATP or an endothelium-derived hyperpolarizing factor, and nitric oxide (NO). An increased release of NO in response to increases in shear stress causes dilatation of underlying smooth muscle of conduit arteries.

Why is FMD significant?

A decreased FMD reflects endothelial dysfunction and may predict coronary artery disease in susceptible patients. The brachial artery FMD can be measured by ultrasound scan and acts as surrogate for the coronary vasculature which can only be assessed by more invasive methods.

How is FMD related to platelet reactivity?

Another effect of NO released by the endothelium under increased shear stress is decrease of platelet reactivity. Does it follow, therefore, that if decreased FMD determines endothelial dysfunction and decreased NO release, it also determines an increased platelet reactivity?

A study by Shechter, et.al. explores the association between platelet reactivity and brachial artery FMD in controls (without established cardiovascular disease) and in patients with acute myocardial infarction. Platelet reactivity was measured by conventional aggregometry and by the IMPACT-R machine. The study concludes that FMD is inversely correlated to platelet reactivity in both controls and AMI patients. An abstract of the said study can be found here.

One of the risk factors for cardiovascular disease is Diabetes. In fact, there is a 2-4 fold increase in the risk of dying from heart disease if the patient is diabetic. One of the reasons is increased production of thromboxane which is a vasoconstrictor and at the same time promotes platelet aggregation.

Acetylsalicylic acid (Aspirin).

Aspirin, or acetylsalicylic acid is a non-steroidal anti-inflammatory drug that has many effects. It is an anti-pyretic, an analgesic and, an anti-inflam-matory drug. It also acts as an anti-platelet because it irreversibly inhibits the  formation of thromboxane through the cyclooxygenase I pathway.

The American Diabetes Association and the American Heart Association have endorsed the following guidelines: “Aspirin may be used in women over 60 and men over 50 who have diabetes mellitus who have other risk factors for heart attack and stroke.”

A study by Spectre, et.al. showed that twice daily dosing of aspirin improved the laboratory outcomes in high-risk DM II patients. The laboratory parameters used were impedance aggregometry (WBA) and the IMPACT-R (CPA). An abstract of the study can be found here.

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.

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.