Cardiology: Blood and body fluids

Constituents of blood

The primary function of blood is to deliver O2 and energy sources to the tissues and to remove CO2 and waste products.
It contains elements of the defence and immune systems, is important for regulation of temperature, and transports hormones and other signalling molecules between tissues. In a 70‐kg man blood volume is ~5500 mL, or 8% of body weight. Blood consists of plasma and blood cells. If blood is centrifuged, the cells sediment as the packed cell volume (PCV, haematocrit), normally ~45% of total volume (i.e. PCV = 0.45) in men, less in women (Figure 5.1).

Plasma

The plasma volume is ~5% of body weight. It consists of ions in solution and a variety of plasma proteins. Normal ranges for key constituents are shown in Figure 5.2. After clotting, a straw‐coloured fluid called serum remains, from which fibrinogen and other clotting factors have been removed. The relative osmotic pressures of plasma, interstitial and intracellular fluid are critical for maintenance of tissue cell volume and are related to the amount of osmotically active particles (molecules) per litre, or osmolarity (mosmol/L); as plasma is not an ideal fluid (it contains slow diffusing proteins), the term osmolality (mosmol/kg H2O) is often used instead. Plasma osmolality is ~290 mosmol/kg H2O, mostly due to dissolved ions and small diffusible molecules (e.g. glucose and urea). These diffuse easily across capillaries, and the crystalloid osmotic pressure they exert is therefore the same either side of the capillary wall. Proteins do not easily pass through capillary walls and are responsible for the oncotic (or colloidal osmotic) pressure of the plasma. This is much smaller than crystalloid osmotic pressure but is critical for fluid transfer across capillary walls because it differs between plasma and interstitial fluid.
Oncotic pressure is expressed in terms of pressure and in plasma is normally ~25 mmHg. Maintenance of plasma osmolality is vital for regulation of blood volume.

Ionic composition

Na+ is the most prevalent ion in plasma and the main determinant of plasma osmolality. Figure 5.2 shows concentrations of the major ions; others are present in smaller amounts. Changes in ionic concentration can have major consequences for excitable tissues (e.g. K+, Ca2+). Whereas Na+, K+ and Cl− completely dissociate in plasma, Ca2+ and Mg2+ are partly bound to plasma proteins, so that free concentration is ~50% of the total.

Proteins

Normal total plasma protein concentration is 65–83 g/L. Most plasma proteins other than γ‐globulins (see below) are synthesized in the liver. Proteins can ionize as either acids or bases because they have both NH2 and COOH groups. At pH 7.4 they are mostly in the anionic (acidic) form. Their ability to accept or donate H+ means they can act as buffers and account for ~15% of the buffering capacity of blood. Plasma proteins have important transport functions. They bind with many hormones (e.g. cortisol, thyroxine), metals (e.g. iron) and drugs and therefore modulate their free concentration and thus biological activity. Plasma proteins encompass albumin, fibrinogen and globulins (Figure 5.3). Globulins are further classified as α‐, β‐ and γ‐globulins. β‐globulins include transferrin (iron transport), components of complement (immune system), and prothrombin and plasminogen, which with fibrinogen are involved in blood clotting. The most important γ‐globulins are the immunoglobulins (e.g. IgG, IgE, IgM).

Blood cells

In the adult, all blood cells are produced in the red bone marrow, although in the foetus, and following bone marrow damage in the adult, they are also produced in the liver and spleen. The marrow contains a small number of uncommitted stem cells, which differentiate into specific committed stem cells for each blood cell type. Platelets are not true cells, but small (~3 μm) vesicle‐like structures formed from megakaryocytes in the bone marrow, containing clearly visible dense granules. Platelets play a key role in haemostasis and have a lifespan of ~4 days.

Erythrocytes

Erythrocytes (red cells) are by far the most numerous cells in the blood (Figure 5.4), with ~5.5 × 1012/L in males (red cell count, RCC). Erythrocytes are biconcave discs with no nucleus, and a mean cell volume (MCV) of ~85 fL. Each contains ~30 pg haemoglobin (mean cell haemoglobin, MCH), which is responsible for carriage of O2 and plays an important part in acid–base buffering. Blood contains ~160 g/L (male) and ~140 g/L (female) haemoglobin. The shape and flexibility of erythrocytes allows them to deform easily and pass through the capillaries. When blood is allowed to stand in the presence of anticoagulant, the cells slowly sediment (erythrocyte sedimentation rate, ESR). The ESR is increased when cells stack together (form rouleaux), and in pregnancy and inflammatory disease, and decreased by low plasma fibrinogen. Erythrocytes have an average lifespan of 120 days.

Leucocytes (white cells) and platelets

Leucocytes defend the body against infection by foreign material. The normal white blood cell count (WBCC, see Figure 5.5) increases greatly in disease (leucocytosis). In the newborn infant the WBCC is ~20 × 109/L. Three main types are present in blood: granulocytes (polymorphonuclear leucocytes, PMN), lymphocytes and monocytes. Granulocytes are further classified as neutrophils (containing neutral‐staining granules), eosinophils (acid‐staining granules) and basophils (basic‐staining granules).
All contribute to inflammation by releasing mediators (cytokines) when activated.
Neutrophils have a key role in the innate immune system and migrate to areas of infection (chemotaxis) within minutes, where they destroy bacteria by phagocytosis. They are a major constituent of pus. They have a half‐life of ~6 h in blood, days in tissue.
Eosinophils are less motile and longer lived and phagocytose larger parasites. They increase in allergic reactions and contribute to allergic disease (e.g. asthma) by release of pro‐inflammatory cytokines. Basophils release histamine and heparin as part of the inflammatory response and are similar to tissue mast cells.
Lymphocytes originate in the marrow but mature in the lymph nodes, thymus and spleen before returning to the circulation. Most remain in the lymphatic system. Lymphocytes are critical components of the immune system and are of three main forms: B cells which produce immunoglobulins (antibodies), T cells which coordinate the immune response, and natural killer (NK) cells which kill infected or cancerous cells.
Monocytes are phagocytes with a clear cytoplasm and are larger and longer lived than granulocytes. After formation in the marrow they circulate in the blood for ~72 h before entering the tissues to become macrophages, which unlike granulocytes can also dispose of dead cell debris. Macrophages form the reticuloendothelial system in liver, spleen and lymph nodes.

Erythropoiesis, haemoglobin and anaemia

Erythropoiesis

Erythropoiesis, the formation of red cells (erythrocytes), occurs in the red bone marrow of adults and the liver and spleen of the fetus. It can also occur in the liver and spleen of adults following bone marrow damage. Erythropoiesis is primarily controlled by erythropoietin, a glycoprotein hormone secreted primarily by the kidneys in response to hypoxia; about 10–15% is produced by the liver, the major source for the fetus. Other factors such as corticosteroids
and growth hormones can also stimulate erythropoiesis.
Erythropoiesis begins when uncommitted stem cells commit to the erythrocyte lineage and under the influence of erythropoietin transform into rapidly growing precursor cells (colony forming unit erythroid cells, CFU‐E) and then proerythroblasts (Figure 6.1). These large cells are packed with ribosomes, and it is here that haemoglobin synthesis begins. Development and maturation proceed through early (basophilic), intermediate (polychromatic), and finally late (orthochromatic) erythroblasts (or normoblasts) of decreasing size. As cell division ceases, ribosomal content decreases and haemoglobin increases. The late erythroblast finally loses its nucleus to become a reticulocyte, a young erythrocyte still retaining the vestiges of a ribosomal reticulum. Reticulocytes enter the blood and, as they age, the reticulum disappears and the characteristic biconcave shape develops. About 2 × 10¹¹ erythrocytes are produced from the marrow each day, and normally 1–2% of circulating red cells are reticulocytes. This increases when erythropoiesis is enhanced, for example after haemorrhage or during hypoxia associated with respiratory disease or altitude. This can greatly increase erythrocyte numbers (polycythaemia) and haematocrit. Conversely, erythropoietin levels may fall in kidney disease, chronic inflammation and liver cirrhosis, resulting in anaemia.
Erythrocytes are destroyed by macrophages in the liver and spleen after ~120 days. The spleen also sequesters and eradicates defective erythrocytes. The haem group is split from haemoglobin and converted to biliverdin and then bilirubin. The iron is conserved and recycled via transferrin, an iron transport protein, or stored in ferritin. Bilirubin is a brown–yellow compound that is excreted in the bile. An increased rate of haemoglobin breakdown results in excess bilirubin, which stains the tissues (jaundice).

Haemoglobin

Haemoglobin has four subunits, each containing a polypeptide globin chain and an iron‐containing porphyrin, haem, which are synthesized separately. Haem is synthesized from succinic acid and glycine in the mitochondria and contains one atom of iron in the ferrous state (Fe2+). One molecule of haemoglobin has therefore four atoms of iron and binds four molecules of O2. There are several types of haemoglobin, relating to the globin chains; the haem moiety is unchanged. Adult haemoglobin (Hb A) has two α and two β chains. Fetal haemoglobin (Hb F) has two γ chains in place
of the β chains, and a high affinity for O2. Haemoglobinopathies are due to abnormal haemoglobins.
Sickle cell anaemia occurs in 10% of the Black population and is caused by substitution of a glutamic acid by valine in the β chain; this haemoglobin is called Hb S. At a low Po2 Hb S gels, causing deformation (sickling) of the erythrocyte. The cell is less flexible and prone to fragmentation, and there is an increased rate of breakdown by macrophages. Heterozygous patients with less than 40% Hb S normally have no symptoms (sickle cell trait). Homozygous patients with more than 70% Hb S develop full sickle cell anaemia, with acute episodes of pain resulting from blockage of blood vessels, congestion of liver and spleen with red cells, and leg ulcers.
Thalassaemia involves defective synthesis of α‐ or β‐globin chains. Several genes are involved. In β thalassaemia there are fewer or no β chains available, so α chains bind to γ (Hb F) or δ chains (Hb A2). Thalassaemia major (severe β thalassaemia) causes severe anaemia, and regular transfusions are required, leading to iron overload (haemochromatosis). In heterozygous β thalassaemia minor there are no symptoms, although erythrocytes are microcytic and hypochromic, that is, mean cell volume (MCV), mean cell haemoglobin content (MCH) and mean cell haemoglobin concentration (MCHC) are reduced. In α thalassaemia there are fewer or no α chains. In the latter case haemoglobin does not bind O2, and infants do not survive (hydrops fetalis). When some α chains are present, patients surviving as adults may produce some Hb H (four β chains); this precipitates in the red cells which are then destroyed in the spleen.

Anaemia

Blood loss (e.g. haemorrhage, heavy menstruation) or chronic disease (e.g. infection, tumours, renal failure) may simply reduce the number of erythrocytes. When these have a normal MCV and MCH, this is termed normocytic normochromic anaemia.
Iron deficiency is the most common cause of anemia. The dietary requirement for iron is small, as the body has an efficient recycling system, but is increased with significant blood loss. Women have a higher requirement for dietary iron than men because of menstruation and also during pregnancy. Iron deficiency causes defective haemoglobin formation and a microcytic hypochromic anaemia (reduced MCV and MCH).
Vitamin B12 (cobalamin) and folate are required for maturation of erythroblasts, and deficiencies of either cause megaloblastic anaemia. The erythroblasts are unusually large (megaloblasts), and they mature as erythrocytes with a high MCV and MCH, although MCHC is normal. Erythrocyte numbers are greatly reduced, and rate of destruction increased. Folate deficiency is mostly related to poor diet, particularly in the elderly or poor; folate is commonly given with iron during pregnancy. Alcoholism and some anticonvulsant drugs (e.g. phenytoin) impair folate utilization. Pernicious anaemia is caused by defective absorption of vitamin B12 from the gut, where it is transported as a complex with intrinsic factor produced by the gastric mucosa. Damage to the latter results in pernicious anaemia. B12 deficiency can also occur in strict vegans.
Aplastic anaemia results from aplastic (non‐functional) bone marrow and causes pancytopenia (reduced red, white and platelet cell count). It is dangerous but uncommon. It can be caused by drugs (particularly anticancer), radiation, infections (e.g. viral hepatitis, TB) and pregnancy, where it has a 90% mortality. A rare inherited condition, Fanconi’s anaemia, involves defective stem cell production and differentiation.
Haemolytic anaemia involves an excessive rate of erythrocyte destruction and causes jaundice. Causes include blood transfusion mismatch, haemolytic anaemia of the newborn, abnormal erythrocyte fragility and haemoglobins, and autoimmune, liver and hereditary diseases. In hereditary haemolytic anaemia (familial spherocytosis) erythrocytes are more spheroid and fragile and are rapidly destroyed in the spleen. It is relatively common, affecting 1 in 5000 Caucasians. Jaundice is common at birth but may appear after several years. Aplastic anaemia may occur after infections, and megaloblastic anaemia from folate deficiency as a result of high bone marrow activity. Figure 6.2 shows characteristics of types of anaemia.

Haemostasis

Primary haemostasis

The immediate response to damage of the blood vessel wall is vasoconstriction, which reduces blood flow. This is followed by a sequence of events leading to sealing of the wound by a clot. Collagen in the exposed subendothelial matrix binds von Willebrand factor (vWF), which in turn binds to glycoprotein Ib (GPIb) receptors on platelets, the first stage of platelet adhesion.
This initial tethering promotes binding of platelet integrin α2β1 and GPVI receptors directly to collagen. Binding of receptors initiates activation, partly by increasing intracellular Ca2+. Platelets change shape, put out pseudopodia and make thromboxane A2 (TXA2) via cyclooxygenase (COX). TXA2 releases mediators from platelet dense granules, including serotonin (5‐HT) and adenosine diphosphate (ADP), and from α granules vWF, factor V and agents that promote vascular repair. TXA2 and 5‐HT also promote vasoconstriction. ADP activates more platelets via P2Y12 purinergic receptors, causing activation of fibrinogen (GPIIb/IIIa) receptors and exposure of phospholipid (PLD) on the platelet surface. Plasma fibrinogen binds to GPIIb/IIIa receptors causing the platelets to aggregate (stick together) forming a soft platelet plug (Figure 7.1). This is stabilized with fibrin during clotting. Note that thrombin is also a potent platelet activator.

Formation of the blood clot

The final stage of blood clotting (coagulation) is formation of the clot – a tight mesh of fibrin entrapping platelets and blood cells. The process is complex, involving sequential conversion of proenzymes to active enzymes (factors; e.g. factor X → Xa). The ultimate purpose is to produce a massive burst of thrombin (factor IIa), a protease that cleaves fibrinogen to fibrin. The cell‐based model of clotting (Figure 7.2) has replaced the older extrinsic and intrinsic pathways. Most of the action in this model occurs on the cell surface (hence its name).
The initial phase of clotting is initiated when cells in the subendothelial matrix that bear tissue factor (TF; thromboplastin) are exposed to factor VIIa from plasma. Such cells include fibroblasts and monocytes, but damaged endothelium and circulating cell fragments containing TF (microparticles) can also initiate clotting.
TF forms a complex with factor VIIa (TF:VIIa) which activates factor X (and IX). Factor Xa with its cofactor Va then converts prothrombin (factor II) to thrombin; activation of both factor X and prothrombin require Ca2+. Comparatively little thrombin is produced at this time, but sufficient to initiate the amplification phase. Activity of these processes is normally suppressed by tissue factor pathway inhibitor (TFPI), which inhibits and forms a complex with factor Xa, which then inhibits TF:VIIa; however, the influx of plasma factors after damage overwhelms this suppression.
The amplification phase takes place on platelets (Figure 7.2). Thrombin produced in the initial phase activates further platelets and membrane‐bound factor V which is released from platelet α granules. Factor VIII is normally bound to circulating vWF, which protects it from degradation. Thrombin cleaves factor VIII from vWF and activates it, when it binds to the platelet membrane.
The scene is now set for the propagation phase. Either factor XIa (itself activated by thrombin) or TF:VIIa can activate factor IX, which binds and forms a complex with factor VIIIa on the platelet membrane called tenase; this is a much more powerful activator of factor X than TF:VIIa. Factors Xa and Va then bind to form prothrombinase on the platelet membrane. This process leads to a massive burst of thrombin production, 1000‐fold greater than in the initial phase and localized to activated platelets.
Factor XII (Hageman factor, not shown) is probably of limited significance, as deficiency does not lead to bleeding. It is activated by negative charge on glass and collagen and can activate factor XI.
It may be involved in pathological clotting in the brain.
Thrombin cleaves small fibrinopeptides from fibrinogen to form fibrin monomers (Figure 7.3), which spontaneously polymerize. This polymer is cross‐linked by factor XIIIa (activated by thrombin in the presence of Ca2+) to create a tough network of fibrin fibres and a stable clot. Retraction of entrapped platelets contracts the clot by ~60%, making it tougher and assisting repair by drawing the edges of the wound together.

Inhibitors of haemostasis and fibrinolysis

Inhibitory mechanisms are vital to prevent inappropriate clotting (thrombosis). Prostacyclin (PGI2) and nitric oxide from undamaged endothelium impede platelet adhesion and activation.
Antithrombin inhibits thrombin, factor Xa and IXa/tenase; its activity is strongly potentiated by heparin, a polysaccharide.
Heparan on endothelial cells is similar. TFPI has already been mentioned. Thrombomodulin on endothelial cells binds thrombin and prevents it cleaving fibrinogen; instead, it activates protein C (APC) which with its cofactor protein S inactivates cofactors Va and VIIIa, and hence tenase and prothrombinase (Figure 7.4).
Fibrinolysis is the process by which a clot is broken down by plasmin, a protease (Figure 7.4). This creates soluble fibrin degradation products (FDPs) including small D‐dimers. Plasmin is formed from fibrin‐bound plasminogen by tissue plasminogen activator (tPA), released from damaged endothelial cells in response to bradykinin, thrombin and kallikrein. Urokinase (uPA) is similar. APC inactivates an inhibitor of plasminogen activator inhibitor (tPA; PAI‐1 and 2), and so promotes fibrinolysis (Figure 7.4). Plasmin is itself inactivated by α2‐antiplasmin and inhibited by thrombin activated fibrinolysis inhibitor (TAFI).

Defects in haemostasis

The most common hereditary disorder is haemophilia A, a deficiency of factor VIII sex linked to males. Christmas disease is a deficiency of factor IX, and von Willebrand disease a deficiency of vWF. The latter leads to defective platelet adhesion and reduced availability of factor VIII, which is stabilized by vWF. The liver requires vitamin K for correct synthesis of prothrombin and factors VII, IX and X. As vitamin K is obtained from intestinal bacteria and food, disorders of fat absorption or liver disease can result in deficiency and defective clotting. Factor V Leiden is brought about by a mutant factor V that cannot be inactivated by APC. Five per cent carry the gene, which causes a fivefold increase in the risk of thrombosis. Antiphospholipid syndrome is caused by phospholipid‐binding antibodies (e.g. cardiolipin, lupus anticoagulant) which may inhibit APC and protein S or facilitate cleavage of prothrombin.
It is associated with recurrent thrombosis and linked to 20% of strokes in people under 50 years, more common in females.

Thrombosis and anticoagulants

Thrombosis

Thrombosis and embolism are ultimately the main cause of death in the industrialized world. Thrombosis is inappropriate activation of haemostasis, with clots (thrombi) forming inside blood vessels.
If thrombi fragment they can be carried in the blood as emboli and block downstream blood vessels causing infarction. Most commonly, fatalities are due to thrombosis as a result of atherosclerotic plaque rupture in acute coronary syndromes, or venous thromboembolism (VTE), particularly pulmonary embolism, following deep vein thrombosis (DVT). Virchow’s triad of endothelial damage, blood stasis and hypercoagulability predisposes to thrombosis. Endothelial (or endocardial) damage is the most common cause of arterial thrombosis. Stasis (poor flow), which allows clotting factors to accumulate and unimpeded formation of thrombi, is the most common cause of DVT and VTE. Risk factors are shown in Figure 8.1. Once formed, thrombi can undergo dissolution by fibrinolysis, propagation by accumulation of more fibrin and platelets, or organization with invasion of endothelial or smooth muscle cells and fibrosis. In recanalization channels form allowing blood to reflow. If not destroyed, thrombi may be incorporated into the vessel wall.
Arterial (white, platelet‐rich) thrombi are primarily treated with antiplatelet drugs, whereas venous (red) thrombi are primarily treated with anticoagulants. All such therapies increase risk of bleeding and may be contraindicated in patients with prior stroke, active ulcers, pregnancy or recent surgery.

Antiplatelet drugs

Aspirin (acetylsalicylic acid) is the most important antiplatelet drug. It irreversibly inhibits cyclooxygenase (COX), the first enzyme in the sequence leading to formation of thromboxane A2 (TXA2) and prostacyclin (PGI2). TXA2 is produced by platelets and is a key platelet activator, whereas endothelium-derived PGI2 inhibits platelet activation and aggregation. Because aspirin inhibits COX irreversibly, production of PGI2 and TXA2 recovers only when new COX is produced via gene transcription.
This cannot occur in platelets, which lack nuclei, whereas endothelial cells make new COX within hours. Aspirin therapy therefore produces a sustained increase in the PGI2: TXA2 ratio, suppressing platelet activation and aggregation. Aspirin can cause gastrointestinal bleeding.
Thienopyridine derivatives such as clopidogrel indirectly and irreversibly block purinergic P2Y receptors, and thus ADP-induced platelet activation; however, they are prodrugs that require metabolism in the liver and so take >24 h for maximal effect. They are useful for aspirin‐intolerant patients and preventing thrombi on coronary artery stents, and long‐term treatment with clopidogrel plus aspirin is beneficial in acute coronary syndromes. Direct P2Y12 receptor antagonists such as ticagrelor and elinogrel have advantages, including reversibility and rapidity of action, and are effective for acute coronary syndrome.
Small peptide glycoprotein receptor inhibitors (GPI) such as tirofiban and eptifibatide and the monoclonal antibody abciximab prevent fibrinogen binding to GPIIb/IIIa receptors on activated platelets, thus inhibiting aggregation. In patients with unstable angina or undergoing high‐risk angioplasty, a GPI combined with aspirin and heparin reduces short‐term mortality and the need for urgent revascularization.

Anticoagulant drugs

Heparin, a mixture of mucopolysaccharides derived from mast cells, activates antithrombin, which inhibits thrombin and factors X, IX and XI. Heparin must bind to both thrombin and antithrombin for inhibition of thrombin, but only antithrombin for inhibition of factor X. Unfractionated heparin has a large variability of action and causes thrombocytopenia in some patients. Low molecular weight heparins (LMWHs) have largely replaced unfractionated heparin in clinical use, as they have a longer half‐life and predictable dose responses; thrombocytopenia is rare. LMWHs bind only to antithrombin and are therefore more effective at inhibiting factor X. They are given subcutaneously and are first-line drugs for routine thromboprophylaxis. Fondaparinux is a synthetic pentasaccharide that acts in a similar fashion to LMWH.
Bivalirudin is a direct thrombin inhibitor delivered intravenously, with benefits of rapidity of action and reversal.
Warfarin (coumarin) is still the most important oral anticoagulant. It inhibits vitamin K reductase and thus γ‐carboxylation of prothrombin and factors VII, IX and X in the liver; this prevents tethering to cells and hence activity. Although slow in onset (~1–2 days), it provides effective support for ~5 days.
Numerous factors including disease and drugs affect the sensitivity to warfarin, so blood tests must be used routinely to monitor dosage, which is adjusted to give a prothrombin time international normalized ratio (INR) of ~3. Use of warfarin may decline following the advent of direct oral anticoagulants (DOACs), for example dabigatran (thrombin antagonist) and rivaroxaban (factor Xa antagonist). These have benefits of increased rapidity of action and reduced sensitivity to other drugs and disease and a greatly reduced need for routine blood tests. Both are approved for prevention of VTE following hip and knee replacement surgery and have been shown to be as effective as warfarin for prevention of atrial fibrillation‐associated stroke.
Thrombolytic agents induce fibrinolysis by activating plasmin; tissue plasminogen activator (tPA) is the most important endogenous agent, and recombinant tPA the most commonly used clinically. Until relatively recently, thrombolysis was the recognized treatment for dissolution of life‐threatening blood clots in coronary artery disease and acute MI, although with a severe risk of gut and intracerebral haemorrhage (stroke). It has now been largely replaced by emergency angioplasty – percutaneous coronary intervention (PCI).

Some laboratory investigations

Prothrombin time (PT): time to clot formation following addition of thromboplastin (TF) (fibrinogen and Ca2+ in excess); normally ~14 s. A measure of activity of vitamin K‐dependent clotting factors and thus important for titrating dose of warfarin. It is expressed as INR, the ratio of the patient’s PT to that of a standardized reference sample. INR is normally 1.
Activated partial thromboplastin time (aPTT): time to clot formation following addition of a surface activator (kaolin; activates factor XII), phospholipid and Ca2+ to plasma. Measures activity of factors in the amplification phase (i.e. not factor VIIa).
Normally 35–45 s. Prolonged by relevant deficiencies.
D‐dimers and fibrin degradation products (FDPs): indicative of fibrinolysis; raised in disseminated intravascular coagulation (DIC) and other thrombotic conditions. False positives common.

Blood groups and transfusions

Blood groups

If samples of blood from different individuals are mixed together, some combinations result in red cells sticking together as clumps (Figure 9.1). This is called agglutination, and it occurs when the blood groups are incompatible. It is caused when antigens (or agglutinogens) on the red cell membrane react with specific antibodies (or agglutinins) in the plasma. If the quantity (or titre) of antibodies is sufficiently high, they bind to their antigens on several red cells and glue the cells together, which then rupture (haemolyse). If this occurs following a blood transfusion it can lead to anaemia and other serious complications. The most important blood groups are the ABO system and Rh (Rhesus) groups.

The ABO system

The ABO system consists of four blood groups: A, B, AB and O. The precise group depends on the presence or absence of two antigens, A and B, on the red cells, and their respective antibodies, α and β, in the plasma (Figure 9.2). The A and B antigens on red cells are mostly glycolipids that differ in respect of their terminal sugar. The antigens are also found as glycoproteins in other tissues, including salivary glands, pancreas, lungs and testes, and in saliva and semen.
Group A blood contains the A antigen and β antibody, and group B the B antigen and α antibody. Group AB has both A and B antigens, but neither antibody. Group O blood contains neither antigen, but both α and β antibodies. Group A blood cannot therefore be transfused into people of group B, or vice versa, because antibodies in the recipient react with their respective antigens on the donor red cells and cause agglutination (Figure 9.3). As people of group AB have neither α nor β antibodies in the plasma, they can be transfused with blood from any group, and are called universal recipients. Group O red cells have neither antigen and can therefore be transfused into any patient. People of group O are therefore called universal donors. Although group O blood contains both antibodies, this can normally be disregarded as they are diluted during transfusion and are bound and neutralized by free A or B antigens in the recipient’s plasma. If large or repeated transfusions are required, blood of the same group is used.

Inheritance of ABO blood groups

The expression of A and B antigens is determined genetically. A and B allelomorphs (alternative gene types) are dominant and O recessive. Therefore AO (heterozygous) and AA (homozygous) genotypes both have group A phenotypes. An AB genotype produces both antigens and is thus group AB. The proportion of each blood group varies according to race (Figure 9.4), although group O is most common (35–50%). Native Americans are almost exclusively group O.

Rh groups

In ~85% of the population the red cells have a D antigen on the membrane (Figure 9.5). Such people are called Rh+ (Rhesus positive), while those who lack the antigen are Rh– (Rhesus negative).
Unlike ABO antigens, the D antigen is not found in other tissues.
The antibody to D antigen (anti‐D agglutinin) is not normally found in the plasma of Rh− individuals, but sensitization and subsequent antibody production occur if a relatively small amount of Rh+ blood is introduced. This can result from transfusion, or when an Rh− mother has an Rh+ child, and fetal red blood cells enter the maternal circulation during birth. Occasionally, fetal cells may cross the placenta earlier in the pregnancy.

Inheritance of Rh groups

The gene corresponding to the D antigen is also called D and is dominant. When D is absent from the chromosome, its place is taken by the allelomorph of D called d, which is recessive.
Individuals who are homozygous and heterozygous for D will be Rh+. About 50% of the population are heterozygous for D, and ~35% homozygous. Blood typing for Rh groups is routinely performed for prospective parents to determine the likelihood of haemolytic disease in the offspring.

Haemolytic disease of the newborn

Most pregnancies with Rh– mothers and Rh+ fetuses are normal, but in some cases a severe reaction occurs. Anti‐D antibody in the mother’s blood can cross the placenta and agglutinate fetal red cells expressing D antigen. The titre of antibody is generally too low to be of consequence during a first pregnancy with a Rh+ fetus, but it can be dangerously increased during subsequent pregnancies, or if the mother was previously sensitized with Rh+ blood. Agglutination of the fetal red cells and consequent haemolysis can result in anaemia and other complications. This is known as haemolytic disease of the newborn or erythroblastosis fetalis. The haemoglobin released is broken down to bilirubin, which in excess results in jaundice (yellow staining of the tissues). If the degree of agglutination and anaemia is severe, the fetus develops severe jaundice and is grossly oedematous (hydrops fetalis), and often dies in utero or shortly after birth.
Prevention and treatment In previously unsensitized mothers, sensitization can be prevented by treatment with anti‐D immunoglobulin after birth. This destroys any fetal Rh+ red cells in the maternal circulation before sensitization of the mother can occur. If haemolytic disease is evident in the fetus or newborn, the Rh+ blood can be replaced by Rh− blood immediately after birth.
By the time the newborn infant has regenerated its own Rh+ red cells, the anti‐D antibody from the mother will have been reduced to safe levels. Phototherapy is commonly used for jaundice, as light converts bilirubin to a more rapidly eliminated compound.

Other blood groups

Although there are other blood groups, these are of little clinical importance, as humans rarely develop antibodies to the respective antigens. However, they may be of importance in medicolegal situations, such as determination of paternity. An example is the MN group, which is a product of two genes (M and N). A person can therefore be MM, MN or NN, each genome coming from one parent.
As with the other groups, analysis of the respective parties’ genomes can only determine that the man is not the father. This method has been largely superseded by DNA profiling.

Complications of blood transfusions

Blood type incompatibility When the recipient of a blood transfusion has a significant plasma titre of α, β or anti‐D antibodies, donor red cells expressing the respective antigen will rapidly agglutinate and haemolyse (haemolytic transfusion reaction). If the subsequent accumulation of bilirubin is sufficiently large, haemolytic jaundice develops. In severe cases renal failure may develop. Antibodies in the donor blood are rarely problematical, as they are diluted and removed in the recipient.
Transmission of infection as a result of bacteria, viruses and parasites. Most important are hepatitis, HIV, prions and in endemic areas parasites such as malaria.
Iron overload resulting from frequent transfusions and breakdown of red cells (transfusion haemosiderosis), for example in thalassaemia. Can cause damage to heart, liver, pancreas and glands. Treatment: iron chelators and vitamin C.
Fever resulting from an immune response to transfused leucocytes which release pyrogens. Relatively common but mild in patients who have previously been transfused and in pregnancy.
Electrolyte changes and suppression of haemostasis following massive transfusions (e.g. major surgery) with stored blood.

Blood storage

Blood is stored for transfusions at 4°C in the presence of an agent that chelates free Ca2+ to prevent clotting; for example, citrate, oxalate and ethylenediaminetetraacetic acid (EDTA). Even under these conditions the red cells deteriorate, although they last much longer in the presence of glucose, which provides a metabolic substrate. The cell membrane Na+ pump works more slowly in the cold, with the result that Na+ enters the cell, and K+ leaves.
This causes water to move into the cell so that it swells and becomes more spherocytic. On prolonged storage the cells become fragile, and haemolyse (fragment) easily. Neither leucocytes nor platelets survive storage well and disappear within a day of transfusion.
Blood banks normally remove all the donor agglutinins (antibodies), although for small transfusions these would be sufficiently diluted to be of no threat. Great care is taken to screen potential donors for blood‐borne diseases (e.g. hepatitis, HIV).