Blood, coagulation and transfusion

Blood cells and plasma

Red blood cells (RBCs, or erythrocytes) typically survive for about 120 days after their release into the circulation. They are created in bone marrow and are released as reticulocytes, which mature over 2 days into adult RBCs. In healthy adults, 1%–2% of RBCs present in the circulation are reticulocytes. Reticulocytes and RBCs do not have nuclei, but residual RNA can still be found in reticulocytes as they mature into erythrocytes.

The classic shape of an erythrocyte is a biconcave disk 8 µm in diameter, but because they deform easily, erythrocytes can pass through capillaries smaller than this.

At the end of their 120-day lifespan, old red cells are destroyed by macrophages in the liver, spleen and bone marrow. The iron contained within is made available for further RBC production, whilst the porphyrins are converted into unconjugated bilirubin.

The primary function of RBCs is to carry oxygen, bound to haemoglobin, to body tissues. In adults the majority of haemoglobin present is HbA (comprising two α- and two β-globin chains: α ₂ β ₂ ). A small amount of HbA ₂ is also present (α ₂ δ ₂ ), as is an even smaller amount of fetal haemoglobin, HbF (α ₂ γ ₂ ). Fetal haemoglobin and HbA ₂ typically represent less than 4% of the total amount of haemoglobin. Each globin chain contains a pocket of haem in which iron is held in its ferrous state, allowing it to bind reversibly with oxygen. As oxygen binds to each haem pocket in turn, the whole haemoglobin molecule changes shape, increasing its overall affinity for oxygen. When the haemoglobin molecule unloads oxygen, the overall affinity for oxygen decreases because 2,3-diphosphoglycerate (2,3-DPG) displaces the two β chains. These changes account for the sigmoid shape of the oxygen–haemoglobin dissociation curve. Increased concentrations of carbon dioxide, hydrogen ions, 2,3-DPG, and sickle haemoglobin (HbS) shift the oxygen–haemoglobin dissociation curve to the right. Fetal haemoglobin does not bind with 2,3-DPG and so its dissociation curve is shifted to the left.

White blood cells (leukocytes) present in the circulation include granulocytes (neutrophils, eosinophils, basophils), lymphocytes and monocytes. The main purpose of white cells is to defend against infection from micro-organisms, and to do this they must be able to pass out of the vasculature into the interstitial space. Once present in tissues, monocytes may differentiate into macrophages.

Neutrophils, monocytes and macrophages are the three major phagocytic cells responsible for the destruction of bacteria, fungi or damaged cells. Phagocytic cells respond to foreign substances in three stages: chemotaxis, whereby phagocytes are attracted to sites of inflammation by chemical signals; phagocytosis, which is where the phagocyte ingests the material in question (often aided by a process called opsonisation, in which particles are tagged by immunoglobulins or complement); and destruction, which is achieved by the release of reactive oxygen species within the cell.

Eosinophils are involved in both allergic reactions and the response to parasitic infections. Lymphocytes are subdivided into B cells, T cells and natural killer (NK) cells. B and T cells release immunoglobulins in response to antigens derived from bacteria, viruses and other foreign particles. Many of these antigens are processed and presented to the lymphocytes by specialist macrophages, termed antigen presenting cells (APC). Lymphocytes that recognise specific antigens can proliferate and produce clones of themselves in response to a specific threat. This threat response is effectively memorised by the organism, resulting in an adaptive immune response . Natural killer lymphocytes do not need prior activation by antigens and are therefore part of an innate immune response, which is responsible for identifying tumour cells or cells invaded by some viruses.

Platelets have a lifespan of approximately 5 days and are produced by the natural breaking apart of megakaryocytes to form cell fragments with no nucleus. Their primary role is haemostasis, but they are also involved in the release of mediators such as fibroblast growth factor.

All the cells within the circulation are suspended in plasma: a mixture of water, electrolytes, proteins such as albumin and globulins, various nutrients such as glucose, and clotting factors.

Blood coagulation

The physiology of haemostasis involves a complex interaction among the endothelium, clotting factors and platelets. Normally the subendothelial matrix and tissue factor (TF) are separated from platelets and clotting factors by an intact endothelium. However, blood vessel damage leads to vasospasm, which reduces initial bleeding and slows blood flow, increasing contact time between the blood and the area of injury. Initial haemostasis occurs through the action of platelets. Circulating platelets bind directly to exposed collagen with specific glycoprotein Ia/IIa receptors. von Willebrand's factor, released from both endothelium and activated platelets, strengthens this adhesion. Platelet activation results in a shape change, increasing platelet surface area, allowing the development of extensions which can connect to other platelets (pseudopods). Activated platelets secrete a variety of substances from storage granules, including calcium ions, adenosine diphosphate (ADP), platelet activating factor, von Willebrand's factor, serotonin, factor V and protein S. Activated platelets also undergo a change in a surface receptor, glycoprotein GIIb/IIIa, which allows them to cross-link with fibrinogen. In parallel with all these changes the coagulation pathway is activated, and further platelets adhere and aggregate ( Fig. 14.1 ).

The classical description of coagulation pathways includes an intrinsic pathway and an extrinsic pathway in which clotting factors are designated with Roman numerals (see Fig. 14.1 ). Each pathway consists of a cascade in which a clotting factor is activated and in turn catalyses the activation of another pathway. The intrinsic pathway involves the sequential activation of factors XII, XI and IX. The extrinsic pathway involves the activation of factor VII by TF and is sometimes called the tissue factor pathway . Of the two pathways, the extrinsic pathway is considered to be the more important because abnormal expression of the intrinsic pathway does not necessarily result in abnormal clotting. The intrinsic pathway may have an additional role in the inflammatory response.

Both the intrinsic and extrinsic pathways result in a final common pathway which involves the activation of factor X. Activated factor X in turn converts prothrombin to thrombin (factor II to IIa), which allows the conversion of fibrinogen to fibrin (factor I to Ia). Fibrin then becomes cross-linked to form a clot.

It is important to note that this description of intrinsic and extrinsic pathways is essentially a description of what happens in laboratory in vitro conditions. The in vivo process is much more of an interplay among platelets, circulating factors and the endothelium.

The following steps can be conceptualised (see Fig. 14.1 ):

• Initiation. Damaged cells express TF, which, after activation by binding with circulating factor VIIa, initiates the coagulation process by activating factor IX to factor IXa and factor X to factor Xa. Rapid binding of factor Xa to factor II occurs, producing small amounts of thrombin (factor IIa).

• Amplification. The amount of thrombin produced by these initiation reactions is insufficient to form adequate fibrin, so a series of amplification steps occurs. Activated factors IX, X and VII promote the activation of factor VII bound to TF. Without this step, there are only very small amounts of activated factor VII present. In addition, thrombin generates activated factors V and VIII.

There is a parallel system of anticoagulation, involving antithrombins and proteins C and S, which helps prevent an uncontrolled cascade of thrombosis. Thrombin binds to thrombomodulin on the endothelium. This prevents the procoagulant action of thrombin. In addition, the thrombin–thrombomodulin complex activates protein C. Along with its cofactor, protein S, activated protein C proteolyzes factor Va and factor VIIIa. Factor Va increases the rate of conversion of prothrombin to thrombin, and factor VIIIa is a cofactor in the generation of activated factor X. Inactivation of these two factors therefore leads to a marked reduction in thrombin production. Activated protein C also has effects on endothelial cells and leukocytes independent of its anticoagulant properties, including anti-inflammatory properties, reduction of leukocyte adhesion and chemotaxis and inhibition of apoptosis.

Antithrombin is a serine protease inhibitor found in high concentrations in plasma. It inhibits the action of activated factors Xa, iXa, XIa, XIIa and thrombin and also factor VIIa from the extrinsic (tissue factor) pathway. Heparin binds to antithrombin to increase the inactivation of thrombin by a factor of more than 2000. Antithrombin deficiency states predispose to thrombosis.

In addition, platelet adhesion and aggregation are normally inhibited in intact blood vessels by the negative charge present on the endothelium, which prevents platelet adhesion, and by substances which inhibit aggregation, such as nitric oxide and prostacyclin.

Controlled fibrinolysis occurs naturally, involving the conversion of plasminogen to plasmin, which in turn degrades fibrin. Plasminogen can be activated by a naturally occurring tissue plasminogen activator and urokinase.

Common laboratory tests used to investigate coagulation include the following:

• Activated prothrombin time (PT), which tests for factors involved in the extrinsic coagulation pathway (prothrombin, factors V, VII, X); normal range 12–14 s, but often expressed as a ratio (the international normalised ratio, INR)

• Activated partial thromboplastin time (APTT, also known as the kaolin cephalin clotting time, KCCT), which tests for factors present within the intrinsic pathway (including factors I, II, V, VIII, IX and X); normal range 26–33.5 s, often also expressed as a ratio (APTTR)

• Thromboplastin time (TT), which tests for the presence of fibrinogen and the function of platelets; normal range 14–16 s

• Fibrinogen assay; normal range 1.5–4 g L ^–1

Monitoring of coagulation

If a patient presents with a known condition or with a history of abnormal bleeding (e.g. menorrhagia or excessive bleeding after previous minor injuries), a coagulation profile including platelet count, PT, TT and APTT is indicated. A platelet count is part of the full blood count considered routine before major surgery. In contrast, coagulation screens should not be considered routine. They are designed for specific investigation of patients with bleeding disorders, not as screening tests (see Chapters 19 and 20 ). They have a low probability of detecting a clinically important abnormality in the absence of any relevant history. During surgery or major haemorrhage, point-of-care viscoelastic monitors such as the thromboelastograph or rotational thromboelastograph measure blood coagulation at the bedside and help aid decision making about which blood products should be given. Unlike other tests of coagulation, thromboelastography (TEG) and rotational thromboelastometry (ROTEM) assess platelet function, clot strength and fibrinolysis. In TEG a small sample of the patient's blood is gently rotated through an angle of 4 degrees and 25 mins, repeated six times a minute to imitate sluggish venous blood flow. A pin is suspended from a torsion wire into the blood sample. Development of fibrin strands couple the motion of the cup to the pin. This coupling is directly proportional to the clot strength. Increased tension in the wire is detected by the electromagnetic transducer, and the electrical signal is amplified to create a trace. From the shape of the trace produced, various measurements indicate the time to clot formation, speed of clot formation, clot strength and fibrinolysis ( Fig. 14.2 ). These values can be used to help decision making about which blood products or antifibrinolytics are required to correct coagulation.

Four values are produced that represent clot formation. The R value represents the time until the clot is first detected. K value is the time from the end of R until the clot reaches 20 mm (speed of clot formation). Alpha angle gives similar information to K. Maximum amplitude (MA) reflects clot strength. LY30 is the percentage decrease in amplitude 30 after MA; it reflects the degree of fibrinolysis. A prolonged R time is usually treated with fresh frozen plasma. The α-angle represents the conversion of fibrinogen to fibrin, and therefore a depressed α-angle is often treated with cryoprecipitate. Most of the MA is derived from platelet function, and therefore a reduced MA is usually treated with platelet transfusions or drugs that improve platelet function such as desmopressin (DDAVP). Rotational thromboelastometry uses a modification of TEG. A disposable pin is attached to a shaft which is connected to a thin spring and slowly oscillates back and forth. Movement originates from the pin and not the cup, and the signal is transmitted using an optical detector instead of a torsion wire. Because of its design, ROTEM is more robust, and it is relatively insensitive to mechanical shocks or vibrations. By using differing activators and inhibitors it is possible to obtain differential information about specific aspects of the coagulation process such as heparin, platelets, coagulation factors, fibrinogen and fibrinolysis. The advantage of these monitors over formal laboratory tests of coagulation is the speed at which the results are obtained, allowing correction of coagulopathies more promptly and with the correct products. Regular calibration, correct use and understanding of results are key to their safe and effective use.