Impact-R, a cone and platelet analyzer can be used to determine the effect of increased erythrocyte and thrombocyte counts on platelet adhesion and aggregation.

In a study by Peerschke, et.al., the cone and platelet analyzer was used to determine platelet adhesion in terms of surface coverage (%SC) and platelet aggregation in terms of aggregate size (AS in um).

A blood clot on electron microscopy.

The study noted that erythrocytosis increased aggregate size and thrombocytosis increased surface coverage. The specimen used was whole blood from healthy individuals with varying RBC and platelet counts.

The study also investigated blood from individuals with myeloproliferative diseases. They noted that differences in the platelet function parameters were seen in MPD patients undergoing different therapies.

Impact-R, a cone and plate analyzer can detect defects in platelet adhesion and aggregation. It can be of use in research about DIC.

What is Disseminated Intravascular Coagulation?

DIC is pathological activation of coagulation mechanisms in response to a variety of substances seen in disease states. Activation of coagulation causes blood clots to form in the small blood vessels of most organs in the body. This has two effects: disruption of blood flow to the organs whose blood vessels have small blood clots resulting in malfunction of the organ; and consumption of blood clotting factors and platelets resulting in bleeding elsewhere in the body.

Normally, the human body is maintained in homeostasis, a balance of coagulation and fibrinolysis, with both processes counterchecking each other through feedback mechanisms. In DIC, these mechanisms are dysregulated. A critical component in the triggering mechanism is tissue factor or TF, also known as platelet tissue factor or Factor III. It is found in subendothelial tissue, platelets and leukocytes. TF is released in response to cytokines, tumor necrosis factor, and endotoxin.

DIC can be caused by many conditions. These include malignancies, obstetrics, trauma, infections, toxins and poisons. These conditions trigger the release of tissue factor and subsequently activate the extrinsic pathway of coagulation.

The role of Impact-R in Research

Since tissue factor can be found in platelets, addition of substances that release tissue factor to platelets will result in coagulation. Impact-R can be used to determine which substances can elicit release of tissue factor.

Impact-R is a cone and plate analyzer which tests platelet function under arterial flow conditions. It can detect defects in platelet adhesion, extension and aggregation such as can be seen in platelet storage pool diseases like a-SPD and s-SPD.

What is d-SPD?

Platelet.

Storage pool disease involving the dense granules was first described in 1972. On Wright-stained smears, the platelets are morphologically normal but are shown to lack d-granules on EM (electron microscopy).  Since these granules are the storage sites for serotonin, ADP and ATP, their lack also means a lack of the said substances.  ADP and ATP are enhancers of platelet aggregation by activation of more platelets which in turn release their dense granule substances and recruit more platelets. Low levels of platelet ADP, ATP result in bleeding diathesis. Examples of bleeding are: easy bruising, epistaxis, post-surgical bleeding, heavy menstrual bleed.

Use of Impact-R in the Diagnosis of d-SPD

Recent studies of platelet adhesion under high-shear stress have been done on patients with d-SPD. One of these is the Impact-R machine. They show a reduced secondary wave of aggregation when stimulated by ADP, epinephrine or thrombin.

Impact-R, a test for platelet function, has potential use as adjunctive diagnostic test to identify protein C or S deficiency.

A blood clot.

What is protein C or S deficiency?

Protein C and S are proteins found in the plasma that participate in the coagulation cascade as regulators of thrombin formation by inhibition of Factor VIIIa and Factor Va. Protein C is activated by thrombin-thrombomodulin complex then combines with free Protein S, its cofactor, to inhibit Factor VIIIa and Factor Va.

Inherited protein C or S deficiency is rare and occurs independently of each other. Protein C or S abnormalities include decreased levels of either protein, inability to bind with each other, protein C/S complex that that cannot degrade factors VIIIa and Va, increased clearance of Protein S.

Deficiencies of either protein C or S results in non-inhibition of factors VIIIa and Va and continuous, unchecked formation of thrombin. This leads to hypercoagulability of patient’s blood, and may result in deep vein thrombosis, venous thromboembolism, purpura fulminans or disseminated intravascular coagulation.

What are the tests for protein C or S deficiency?

Laboratory tests include Functional Protein C, Functional Protein S, Protein C Antigen and Protein S Antigen. These are specific for the Protein deificiency.

Adjunctive tests include Bleeding time, Partial Thromboplastin time, Prothrombin time and Thrombin time.

What is the use of Impact-R in diagnosing protein C or S deficiency?

Impact-R can be of use in the diagnosis of protein C or S by determining the effect of adding free protein S and activated protein C in the platelet adhesion and aggregation of whole blood of patients with the suspected deficiency. This can be an alternative in cases where the Protein C or S assays are not available.

Impact-R, a cone and plate analyzer for platelet adhesion and platelet aggregation may be utilised as a screening test for bleeding disorders such as Hemophilia A.

What is Hemophilia A?

Hemophilia A is a blood disorder that causes prolonged bleeding due to a deficiency of blood clotting Factor VIII.

Hemarthrosis of the elbow joint.

Symptoms are spontaneous bleeding, bruising, blood in the urine, gastrointestinal bleeding, epistaxis, prolonged bleeding from cuts and following surgeries such as circumcision and dental extraction, hemarthroses or bleeding into joints such as the knee. Symptoms may not be apparent in infants but will soon show when the baby starts to crawl or walk. Severity of the symptoms may vary. Major bleeding could be fatal such as those that happen if the patient is involved in a motor vehicular accident or sports-related injuries.

Hemophilia A is a hereditary disorder that is X-linked recessive. The disease manifests only in males because of their single X chromosome, whereas, females have two X-chromosomes. Very rarely, females manifest the disease if both X chromosomes are affected. This happens when the father is a haemophiliac and the mother is a carrier. Affected males mated to non-carriers will have male offspring that do not have the disease and female offspring that are carriers. Carrier females mated to un-affected males will have 50% chance of having male children with the disease and 50% chance of having female children who will be carriers.

The Use of Impact-R

Impact-R could be of use as a bedside screening test in children whose mothers have male relatives that have Hemophilia. It is a simple and rapid test that can determine defects in coagulation affecting platelet function. Studies have to be done, however, to determine the positive and negative predictive value of Impact-R as a screening test for Hemophilia A.

The Impact-R machine provides high-shear stress conditions needed by ADAMTS-13 to cleave ULVWF multimers seen in Thrombotic Thombocytopenic Purpura.

Skin lesions in TTP.

Thrombotic thrombocytopenic purpura is a rare disorder of the coagulation cascade. The etiology of TTP includes that of inhibition of function or the deficiency of ADAMTS-13, a metalloprotease enzyme that cleaves von Willebrand factor multimers in high shear stress conditions. If it is inhibited or deficient, the vWF forms ultra large multimers that predispose to thrombi formation in the microvasculature by spontaneously attaching to the platelet membrane GpIba.

A study by Jing Fei Dong, et.al.demonstrates this phenomenon. In the study,

Beads-on-a-string formation of platelets and ULVWF multimers.

cells with GpIba receptors in their membranes and platelets formed long beads-on-a-string formations on the endothelial culture cells when exposed to ultra large von Willebrand factor (ULVWF) multimers under high shear stress conditions.

When blood from a patient with TTP is tested using the Impact-R, surface coverage and aggregate size are expected to be large because of the presence of ULVWF in the patient’s plasma. When the same blood is exposed to plasma from a normal patient (which contains ADAMTS-13), and then tested by the Impact-R, theoretically, there will be a lessening of the surface coverage and aggregate size. This is the subject of a study by Shenkman in which he proposed that Impact-R could be used as a rapid screening test for TTP.

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.