Platelet Plug and Fibrin Clot Formation - Essay Example

? Haematology - Haemostasis Vascular injury initiates various multistep processes to ensure that a clot is formed at the site of injury to prevent any loss of blood. Two important steps involved in the prevention of blood loss and repair include platelet plug and fibrin clot formation. Initial damage to the endothelial wall promotes the expression of von Willebrand factor (vWF) on the damaged cells at the site of injury. These factors acts like a glue to adhere the fast moving platelets with the strength sufficient enough to withstand the streamline force of the blood. There are certain glycoproteins present on the platelet surface that takes part in platelet adhesion and aggregation. Also the collagen receptors present on the platelet can directly bind to subendothelial collagen further anchoring the adhesion. After initial adhesion, platelets are activated releasing factors that promote platelet aggregation. This aggregation occurs mostly by inter platelet interaction of Gp IIb/IIIa receptors present on the surface membrane. All these events lead to platelet plug formation. (Harrison 2005, p337) To stabilize the initial platelet plug, fibrin meshwork is required which only becomes available after series of activation of various clotting factors. The activation of these clotting factors is like a cascade reaction in which one activates the other until a loop is formed. There are two distinct pathways; extrinsic and intrinsic pathways. Tissue factor (TF) initiates coagulation by activating classic extrinsic pathway, whereas, intrinsic pathway play a role in propagation of the cascade. All this activation takes place mostly at the phospholipid membranes of the platelets. Tissue factor responsible for initiation is expressed by damaged and exposed components of the vessel wall as well as released by platelets and monocytes. Coagulation process ultimately activates thrombin from its inactive state which in turn converts fibrinogen into fibrin. These fibrins then arrange in an orderly manner, providing a mesh work that anchor the platelets and form a fibrin clot at the site of injury. (Harrison 2005, pp.337-339) 2. Three major components of Virchow’s triad include stasis, hypercoagulability and endothelial injury. This triad basically explains the haemostatic mechanisms that promote thrombus formation. Stasis is the interruption to the normal blood flow or obstruction in the circulatory system. Immobility, venous insufficiency and mitral stenosis are few causes which promote stasis of blood. Heart arrhythmias can also lead to turbulence of blood, interrupting the normal flow. Stasis allows the settlement of clotting factors and platelets, thereby, promoting thrombosis. Muscle contraction is a major force that moves the venous blood towards heart against gravity. Therefore, immobility results in pooling and stasis of blood in the lower extremity and is an important cause of deep venous thrombosis. (Torshin 2007, p55) Damage to vascular endothelium is a very strong promoter of thrombus formation. Thrombus formation at the site of vascular injury is a normal physiological mechanism that aims to prevent any blood loss. Endothelial injury results in platelet adhesion and aggregation with the release of tissue factor that activates the coagulation cascade ultimately forming the fibrin meshwork that anchors the platelets and form a thrombus. Common causes of vascular endothelial injury include long standing hypertension, inflammation and trauma. (Torshin 2007, p55) There are many factors that can predispose to hypercoagulability state such as pregnancy, use of oral contraceptive pills, sepsis, trauma etc. Most importantly any pathology that increase blood viscosity or increase cellular components of plasma can lead to hypercoagulable state. Polycythemia and myeloproliferative disorders are few examples. Similarly factors deficiency of factors that inhibit coagulation can promote thrombus formation. (Torshin 2007, pp.55-56) 3: Three major components of Virchow’s triad include stasis, hypercoagulability and endothelial injury. This triad basically explains the haemostatic mechanisms that promote thrombus formation. Stasis is the interruption to the normal blood flow or obstruction in the circulatory system. Immobility, venous insufficiency and mitral stenosis are few causes which promote stasis of blood. Heart arrhythmias can also lead to turbulence of blood, interrupting the normal flow. Stasis allows the settlement of clotting factors and platelets, thereby, promoting thrombosis. Muscle contraction is a major force that moves the venous blood towards heart against gravity. Therefore, immobility results in pooling and stasis of blood in the lower extremity and is an important cause of deep venous thrombosis. (Torshin 2007, p55) Damage to vascular endothelium is a very strong promoter of thrombus formation. Thrombus formation at the site of vascular injury is a normal physiological mechanism that aims to prevent any blood loss. Endothelial injury results in platelet adhesion and aggregation with the release of tissue factor that activates the coagulation cascade ultimately forming the fibrin meshwork that anchors the platelets and form a thrombus. Common causes of vascular endothelial injury include long standing hypertension, inflammation and trauma. (Torshin 2007, p55) There are many factors that can predispose to hypercoagulability state such as pregnancy, use of oral contraceptive pills, sepsis, trauma etc. Most importantly any pathology that increase blood viscosity or increase cellular components of plasma can lead to hypercoagulable state. Polycythemia and myeloproliferative disorders are few examples. Similarly factors deficiency of factors that inhibit coagulation can promote thrombus formation. (Torshin 2007, pp.55-56) 4. The typical coagulation cascade consists of multiple factors, both enzymatic and non enzymatic protein domains. These non enzymatic protein domains are also called the co-factors because they help in the activation of other enzymatic factors. Tissue factor, factor V, factor VIII and high molecular weight kininogen (HMWK) are all non enzymatic proteins that are found as part of the typical coagulation enzymes. It is important to remember that coagulation cascade is most commonly initiated by tissue factor through activation of classic extrinsic pathway. Tissue factor binds to factor VIIa to activate two other factors, factor IX and X. Factor X after activation ultimately convert prothrombin into thrombin which goes on to cleave profibrogen into fibrin. Thrombin is also responsible for the activation of factor V and factor VIII, two other non enzymatic protein domains. Factor V is an important co factor that takes part in the activation of thrombin. Factor VIII helps in the activation of factor X by forming a complex with factor IX. This pathway normally propagates the cascade but does not take part in its initiation. (Harrison 2005, pp.337-339) High molecular weight kininogen (HMWK) is a co-factor for factor XIIa. So in conclusion these non enzymatic protein domains do not directly perform any enzymatic cleavage for the activation of other coagulation factors but they are still essential for the functioning of typical coagulation enzymes. Transfusion 1: Haemolytic disease of the newborn (HDN), result from alloimmune destruction of foetal RBCs by mother’s IgG antibodies in utero. Red blood cells have various antigens on their surface clinically divided into different system. Rhesus system of antigens has various different types, but RhD antigen is the one most commonly associated with HDN. In a classical scenario, mother is Rh negative, meaning with no D antigen on its red blood cell and the growing foetus do have D antigen and are Rh positive. So when some foetal RBCs leak into maternal circulation, mother’s immune cells recognize this D antigen as foreign and produce antibodies against it which cause hemolysis in foetus. Mother may have been sensitized prior to pregnancy either by transfusion or previous pregnancies. Identifying the blood group of parents is the first step towards diagnosing HDN. This is because if mother is Rh positive, the likelihood of this disease is very less. Moreover, identifying baby’s blood group and identification of Rh D positive type increase the likelihood of HDN. Clinical signs and symptoms such as jaundice and hyperbilirubinemia in baby also indicate a haemolytic problem. But confirmatory diagnosis is based on immuno assays which detect the presence of these alloantibodies. A direct Coombs’ test is performed on foetal RBCs to confirm the presence of anti D antibodies. Similarly an indirect combs’ test can be carried out in mother to further confirm the diagnosis. (Yousuf et al. 2012) Management can be divided into before birth and after birth. If the diagnosis is made before birth, intrauterine transfusion can be performed and labour can be induced as soon as lung maturity is reached to minimize exposure to the maternal antibodies. After birth, blood transfusion, phototherapy for jaundice and intensive care is required for steady recovery of newborn. Rhogam, a drug consisting of immunoglobulins that bind D antigen and prevent maternal sensitization can be administered at specific times to prevent HDN. Therefore, it is important that ABO and Rh blood grouping is performed on regular basis during antenatal checkups to prevent the occurrence of this disease. (Yousuf et al. 2012) 2: Foetal maternal haemorrhage is especially important to diagnose when mother is rhesus antigen negative and foetus is positive because of increased risk of haemolytic disease of newborn. In a normal pregnancy, maternal and foetal blood does not mix and are kept in separate compartments by two placental membranes across which most of the exchange takes place. Normally, a small volume of foetal blood does crossover but in that case maternal sensitization towards Rh D antigen is very slow due to less number of available antigens. In pathological cases, such as placental abruption, mixing of blood can be quiet significant. Laboratory role is to determine if mixing of foetal and maternal blood has taken place and to quantify how much blood is mixed. There are two important tests performed for this purpose which includes Rosette and Kleihauer-Betke test. Rosette test is a qualitative screening test that determines the presence of Rh D positive foetal cells in the maternal blood. To quantify this mixing, mono layer blood films of maternal blood are treated with Kleihauer-Betke stain. With further treatment, this test reveals ghost cells which are actually foetal cells. Calculation of these cells gives a quantitative analysis of foetal maternal mixing. (Rudmann 2005, p441) During routine antenatal check up, lab investigation for the presence of maternal anti D antibodies is critical for prevention of haemolytic disease of the newborn. Tests such as direct or indirect Coombs’ test can also diagnose the presence of this disease. The basic aim of lab investigation is to determine the possible risk as early as possible and to prevent the sensitization of maternal immune system towards foetal rhesus or ABO antigens. Even routine screening of blood before transfusion and accurate matching can help in this process and prevent the occurrence of HDN. 3. The electronic issue of red cells unit is becoming a common practice in most advanced transfusion laboratories. This procedure, cross match without the traditional serological testing system, therefore, strict guidelines issued by British committee for standard in haematguidelines (BCSH) should be followed. These guidelines stress that ABO and RhD group of the patient must be determined twice. Moreover, the blood group of the tested patient, the current sample, and historic blood group should be same. All these testing should be performed using full automation without any manual editing. Anti body screen should be done as per BCSH guidelines and the sample should be negative for antibodies. Patients unsuitable for electronic issue such as those with sickle cell, positive direct antiglobulin test or post marrow or organ transplant patients should be properly flagged. There must be strict IT checks on the computer systems as they should not allow the selection of ABO incompatible red cells. There are certain advantages and disadvantages of electronic issue. The system is fast and more reliable than manual processing but there have been incidences where inappropriate flagging of the patients have resulted in transfusion of electronic issue red cells to few exceptions discussed above. This system of testing is not reliable for transfusing blood to patients who have undergone marrow or other organ transplants. The risk of incorrect determination of ABO and RhD group is a possibility if testing is not fully automated. Therefore, it can lead to serious morbidity and complications. 4. Serious hazard of transfusion (SHOT) scheme is a United Kingdom based organization led by professional in the field of hematological surveillance. Since its foundation in 1996, it has monitored and recorded reports of any adverse events and reactions across the country dissecting the issues and formulating a report with recommendations to improve patient safety. Blood transfusion poses many threats to patients worldwide which varies in severity from mild physiological hemodynamic disturbances to life threatening adverse reactions. According to 2011 SHOT annual report, there were 8 deaths in UK due to transfusion or lack of it with 117 incidences of major morbidity. The main hazards addressed in the report includes transfusion-associated graft versus host disease (TA-GvHD), transfusion-transmitted infections (TTI), transfusion related acute lung injury (TRALI), post transfusion purpura (PTP), wrong transfusions with inappropriate handling and near miss incidences. According to the report wrong transfusion and near miss incidence are unfortunately high as compared to other hazards mentioned earlier. (SHOT annual report, 2012) Blood can serve as an excellent medium for growth and propagation of bacteria and other pathogens. Therefore, transfusion of whole blood or its component is a possible source of transmitting this disease causing pathogens from one individual to the other. As fresh frozen plasma and packed RBCs are stored at very low temperatures, bacterial contamination is less likely but certain bacteria such as Yersinia, Pseudomonas, Serratia, Acinetobacter and Escherichia species can grow at those temperatures as well. Platelets are stored at room temperature so the risk of bacterial contamination is very high. Pre-transfusion screening of blood components, especially platelets, is important to decrease the risk of transfusion induced sepsis. (Harrison 2005, p667) 5: The four components of blood commonly found in blood banks include whole blood, fresh frozen plasma, red cells unit and platelets. Later three components of whole blood are separated by a technique called Apheresis. In this technological era, various sophisticated instruments are available for automated separation of blood components. (Rudmann 2005, p223) After separation of these components, appropriate conditions are required for optimal duration of storage. Apart from adding anticoagulants, other physical factors such as temperature should be closely regulated for the storage of blood. The refrigerators used for this purpose are specially designed with proper labelling of each shelves and digital read out thermometers for constant temperature monitoring. Different components of blood require different temperature settings. For example, temperature of refrigerators containing whole blood and RBC units are typically maintained between 1-6 oC. Whereas, platelets and granulocytes require room temperature for optimum storage so their containers are maintained at temperatures of 20oC to 24oC. Platelets may aggregate if not continuously agitated; therefore, specially designed rotators gently agitate platelets to prevent aggregation and to promote gas exchange. (Rudmann 2005, p223) Whole blood can be given to almost all patients requiring any one or more components of the blood. But if promptly available, only specific components of blood that are specifically required for patients are transfused. Specific cases, in which patient is suffering from anaemia or low haemoglobin levels, RBCs can be selectively transfused. Patients with haemolytic disorders suffering from thrombocytopenia are given platelets while fresh frozen plasma can be administered to hypovolemic patients. Anaemia 1: Macrocytosis simply means increase in cell size which is greater than the normal range. Conditions in which macrocytosis is seen can be classified as either megaloblastic or non megaloblastic macrocytosis. Megaloblastic anaemia is most commonly associated with cobalamin (Vitamin B12) or folate deficiency, whereas, non megaloblastic condition may arise due to alcohol intoxication. It is important to differentiate these two conditions. In the case of cobalamin or folate deficiency, anaemia is a common feature but in non-megaloblastic condition anaemia may or may not be present. To comprehend these differences it is important to understand the basic underlying pathogenesis of these diseases. Both cobalamin and folate play integral roles in various processes of DNA synthesis. Therefore, there deficiencies can impair cellular maturation and division. These effects are commonly seen in rapidly dividing cells such as RBCs, leukocytes, megakaryocytes etc. As these cells are not able to divide, their cytoplasmic contents increase leading to macrocytosis and impaired maturation lead to blastic cells which are basically immature cells. This is the reason why this is called megaloblastic condition. Hypersegmented neutrophils are commonly seen in megaloblastic anaemia but they are absent in non-megaloblastic macrocytosis. This is because the cellular maturation is not impaired in non-megaloblastic condtions. Non-megaloblastic condition is most commonly seen as a result of alcohol intoxication that only affects RBCs. Clinical findings include increase in mean cell volume (MCV) with the presence of macrocytic target cells. (Harrison, 2005) 2. When a patient presents with symptoms of anaemia, such as shortness of breath (mostly during work), fatigue, lethargy, loss of energy and pallor, a series of laboratory work up is required to confirm the diagnosis and to determine the underlying aetiology. As in the case of microcytic anaemia, complete blood count is an essential lab test required for evaluating haemoglobin, hematocrit, and red blood cells indices. Haemoglobin is a protein, present in the red blood cells, responsible for oxygen transport around the body. A decrease in haemoglobin levels indicates presence of anaemia but the aetiology cannot be determined from these levels. Red blood cells indices are important to differentiate macrocytic from microcytic anaemia. Normal range of Mean Corpuscle Volume (MCV) is about 90 +/-8 but it is usually high in macrocytic anaemia and falls below normal in microcytic anaemia. Peripheral blood smear and reticulocyte counts do not provide direct evidence of microcytic anaemia but they can support the diagnosis by ruling out the other aetiologies. Iron deficiency is a most common cause of microcytic anemia, therefore, once microcytic anaemia is suspected further tests for iron supply and storage are required. The parameters tested for this purpose include total iron binding capacity (TIBC), serum iron and percent transferring saturation which is derived by dividing serum iron levels with TIBC. Total iron binding capacity is increased in iron deficiency. Total body iron stores are evaluated by determining serum ferritin levels. A decrease in these levels indicates insufficient or depleted body iron stores. (Harrison 2005, pp.330-332) 3: During red blood cells synthesis, a blast cell undergoes variety of morphological and cellular changes before it matures as an erythrocyte. Haemoglobin is an important component of RBC and its production continues even in the circulating reticulocyte. A haemoglobin molecule is basically composed of four haemoglobin chains and iron is required as a prosthetic group for the synthesis of heme portion of these haemoglobin chains. Two steps pathway synthesizes protoporphyrin IX from succinyl-CoA. Iron is combined with protoporphyrin in the subsequent step to form the heme molecule which is attached to the polypeptide chain to form haemoglobin chains. In a normal healthy state, enough iron is present in the bone marrow or in the reserves for sufficient production of haemoglobin for RBCs. (Guyton & Hall, 2000) There are various pathological states that can result in microcytic red cell indices. Most commonly it is due to iron deficiency that can be associated with chronic blood loss, insufficient dietary intake or abnormally high rate of hemolysis. All these states, except for dietary iron deficiency, will lead to decreased hematocrit or lower number of RBCs in the blood. To compensate this decrease, body will respond by trying to increase the production rate of RBCs utilizing more iron than is absorbed from the intestines. Therefore, over a period of time the iron reserves will deplete ultimately leading to iron deficiency. As iron levels fall, haemoglobin chain synthesis is also impaired because it is required for heme synthesis, as explained earlier. This decrease haemoglobin synthesis will result in abnormally small sized RBCs with decreased mean cell volume (MCV), mean cell haemoglobin (MCH) and mean cell haemoglobin concentration (MCHC). (Harrison 2005, pp.330-331) 4: Vitamin B 12 or cobalamin deficiency is a common cause of macrocytic anaemia. This is because it is required for number of processes involved in DNA synthesis. Therefore, the deficiency will lead to abnormalities associated with maturation of RBCs. Investigation of B12 deficiency is a two stage process. First of all, it is important to confirm the presence of macrocytic anaemia and then in the second step, clinicians can work out if the underlying cause is vitamin B12 or folate deficiency as both present with similar haematological findings. (Harrison 2005, p601) Like in most cases of anaemia, the primary lab investigation is the complete blood count (CBC). The result can indicate the presence of anaemia by measuring the haemoglobin levels and macrocytic component can be ruled in if mean cell volume is elevated. Peripheral blood smear is an important lab test that may show hypersegmented neutrophils with reduction in granulocytes and lymphocytes. Bone marrow and chromosome analysis may also suggest abnormal DNA synthesis and cellular maturation. (Harrison 2005, p605) Once diagnosis of macrocytic anaemia is confirmed, investigation can be targeted to identify the cause of vitamin B12 deficiency. Enzyme-linked immunoadsorbent assay (ELISA) is used to determine the serum cobalamin levels. Normal range is between 118-148 pmol/L but in deficiency states it can go below 74 pmol/L. Cobalamin absorption require an intrinsic factor released from the parietal cells of stomach. Its deficiency as in the case of gastric atrophy, gastrectomy or congenital defect can lead to pernicious anaemia. Therefore, even a normal or excess intake of cobalamin is useless unless intrinsic factor is given along. Malabsorption, as in the case ileal resection, stagnant loop syndrome, tropical sprue and other intestinal manifestations can lead to cobalamin deficiency. Therefore, schilling test is commonly done to rule out intestinal absorption problems and sometime performed along with intrinsic factors to identify or rule out pernicious anaemia. (Harrison 2005, p606) 5 (i): Iron is an important element is required my almost all cells in the body for various functions. But apart from its benefits, it can also be harmful if not handled properly because free iron can catalyze the production of free radicals. So a proper metabolic mechanism is required to ensure its physiological activity and inhibit its toxicity. Iron is absorbed in the proximal small intestine and absorption is facilitated by the acidic content of the stomach. It is important to remember that there is no excretion mechanism for iron in a human body, unless there is a blood loss, so its absorption is tightly regulated. At the brush border of the luminal cell, ferric ions are converted to ferrous state by ferrireductase for absorption across DMT-1 transporter. Once inside the luminal cell, iron can either be stored as ferritin or released into blood plasma for attachment with transferrin, a transport protein for iron. Before iron can ride onto transferrin, its release from luminal cell requires a passage through iron exporter called ‘ferroportin’ which is the regulatory site for hepcidin, a principle iron regulatory hormone. Low levels of this hormone will increase iron absorption and binding with the transferrin and vice versa. Other factors such as food source of iron can also affect its absorption. Heme iron from red meat is more readily absorbed than other vegetarian sources. (Harrison 2005, pp.586-588) 5 (ii): In normal erythropoiesis, iron is required for the formation of heme molecule from succinyl CoA in a three step process. The heme molecule formed is then attached to polypeptide chains to from hemoglobin chains. These chains are actually the precursor of hemoglobin molecule which is essentially required by all RBCs for transporting oxygen. In the case of iron deficiency, there will be shortage of these chains and, therefore, decrease production of hemoglobin. 5 (iii): Decrease cytoplasmic synthesis of hemoglobin chains will result in a small sized RBC and anaemia characterized by such cells are collectively called as microcytic anaemia. The adult red cells indices for such patients will show decreased mean cell volume (MCV) with decreased mean cell hemoglobin (MCH) and mean cell hemoglobin concentration (MCHC). (Harrison 2005, pp.330-331) Reference (2012). SHOT Annual Report 2011. Summary of findings, lessons and recommendations.BIOMEDICAL SCIENTIST. 56, 485-489 GUYTON, A. C., & HALL, J. E. (2000). Textbook of medical physiology. Philadelphia, Saunders. HARRISON, T. R. (2005). Harrison's principles of internal medicine. New York, McGraw-Hill Medical Pub. Division. RUDMANN, S. V. (2005). Textbook of blood banking and transfusion medicine. Philadelphia, Elsevier/Saunders. TORSHIN, I. Y. (2007). Physiology and medicine. New York, Nova Biomedical Books. YOUSUF, RABEYA, ABDUL AZIZ, SURIA, YUSOF, NURASYIKIN, & LEONG, CHOOI-FUN. (2012). Hemolytic disease of the fetus and newborn caused by anti-D and anti-S alloantibodies: a case report. BioMed Central Ltd. BioMed Central Ltd. http://www.jmedicalcasereports.com/content/6/1/71. Read More