Blood and Related Physiology

8/4/2019 Blood and Related Physiology

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Blood and related

physiology

2.1 Blood volume and constituents 37

2.2 Plasma constituents 37

2.3 Erythrocytes 38

2.4 Leucocytes 42

2.5 Immune responses 43

2.6 Platelets and haemostasis 49

Self-assessment: questions 51

Self-assessment: answers 55

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ChapterChapter

packed red cells to the total blood volume is referredto as the haematocrit or packed cell volume (Fig. 22),which is normally about 0.45, or 45%. A thin layer ofwhite cells and platelets can also be identified at theinterface between the red cells and the plasma (the

buffy coat). Plasma comprises 55% of total bloodvolume, i.e. about 3 litres in an adult.

2.2 Plasma constituents

Overview

Blood is circulated around the body within the cardio-vascular system, transporting O2, necessary metabolicsubstrates and hormones to the cells of the body, whileremoving CO2 and waste products. Plasma, the liquidphase of blood, has many functions, involving colloid

osmotic effects, transport, signalling, immunity andclotting. The cellular elements of blood are responsiblefor gas transport, immunity and some aspects ofhaemostasis.

2.1 Blood volume andconstituents

Learning objectives

At the end of this section you should be able to: state the normal values for blood volume, plasmavolume and haematocrit.

Blood volume averages about 70 ml kg1 body weight,giving a total of approximately 5 L in an adult. Thisconsists of a suspension of red cells, white cells andplatelets (theformed elements) inplasma. Centrifuginga sample of blood separates the formed elementsfrom the plasma and the ratio of the volume of the

Learning objectives

At the end of this section you should be able to: classify the plasma proteins and outline their

functions.

Plasma has the same ionic composition as therest of the extracellular fluid (Section 5.1) but alsocontains plasma proteins (total concentration ofabout 60 g L1), with a range of important functions.

Albumin (synthesized by the liver) is the proteinin highest concentration and so it makes thegreatest contribution to the colloid osmoticpressure of plasma (Section 3.8). It also acts as anonspecific transport protein for a number ofsubstances with a low solubility in water, e.g.,

bilirubin. Globulins include specific transport proteins (e.g.,transferrin for iron), clotting factors, thecomplement system and inactive precursors ofcertain hormones, e.g., angiotensinogen. Onesubtype, known as gamma globulins, orimmunoglobulins, acts as circulatingantibodies important in specific immuneresponses.

Fibrinogen is converted to fibrin duringclotting.


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2.3 Erythrocytes

Erythrocyte development

The process of red cell production, or erythropoiesis,begins in the embryonic yolk sac and is continued inthe liver, spleen and lymph nodes in the maturingfetus. By the end of pregnancy and after birth,however, the process is restricted to bone marrow.As time progresses, the contribution from long bones

decreases and in adult life only the marrow of mem-branous bones, such as the vertebrae, ribs and pelvis,is involved.

Stages in erythrocyte developmentPluripotential, or uncommitted, stem cells, whichhave the potential to produce any type of blood cell,divide and develop into erythroid stem cells com-mitted to form erythrocytes (Fig. 23). These dividefurther and mature, synthesizing haemoglobin andeventually forming normoblasts. Nuclear material is

extruded and the endoplasmic reticulum resorbed,producing first a reticulocyte, containing a few rem-nants of endoplasmic reticulum, and then an eryth-rocyte. Normally only these last two cell types arefound in the circulation, with reticulocytes makingup less than 2% of the total. This percentage risesduring periods of rapid erythrocyte synthesis, whenmore immature cells enter the circulation. Maturered cells take the form of biconcave discs whichdeform easily within the narrow capillaries. Thenormal red cell count in blood is 4 1012 to 6 1012 L1

(46 106 mm3).

Erythrocyte destruction

Ageing erythrocytes are destroyed, often in thespleen, after an average life span of 120 days. Thephagocytic cells of the reticuloendothelial systemdegrade the haemoglobin released, with iron fromthe haem and amino acids from the globin molecules

being recycled. The porphyrin ring is converted tobilirubin, which is further metabolized by the liverand then excreted in bile (Section 6.3).

Control of erythrocyte production

Erythropoiesis is controlled by the kidney, whichreleases a hormone known as erythropoietin if thedelivery of O2 to renal cells falls below normal. Thiswill occur if the concentration of circulating haemo-globin is reduced, i.e., during anaemia. The bonemarrow responds by increasing red cell production,thus increasing the haemoglobin content back tonormal. Since this control loop is sensitive to tissueO2 levels rather than the actual haemoglobin concen-tration, other conditions which reduce the O2 content

Fig. 22 Determination of the haematocrit from a centrifuged bloodsample.

Learning objectives

At the end of this section you should be able to: state normal values for [haemoglobin], red cell

count, red cell life span and erythrocytesedimentation rate (ESR)

outline the structure of haemoglobin and its role inO2 transport

outline how red cells are manufactured and

destroyed explain how nutritional deficiencies lead to

anaemia

describe how ABO and Rh blood groups aredetermined and the consequences of blood groupmismatch.

Red blood cells, or erythrocytes, have several func-tions but are particularly important as O2 deliverysystems. This reflects the O2 transport characteristicsof haemoglobin, which is packaged inside erythro-

cytes so that it does not leak out of capillaries. Hae-moglobin consists of four peptide chains (the globinstructure) each linked to a haem molecule consistingof a porphyrin ring structure encircling a ferrousiron ion (Fe2+). The reversible binding of O2 to theseions accounts for 97% of the normal O2-carryingcapacity of blood, so haemoglobin concentrationdetermines O2 transport capacity. Normal haemo-globin values are in the range of 1416 g dl1 in menand 1214 g dl1 in women. A low blood haemoglo-

bin concentration is referred to as anaemia.

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of blood will also stimulate erythropoiesis, evenif the haemoglobin concentration is normal. Thisis seen at high altitudes, where the partial pressuresof O2 in the lungs and blood are reduced(Section 4.6). Over a period of weeks at high alti-tudes, erythropoietin stimulates an increase in thehaemoglobin concentration, with a rise in haemato-crit and red cell count (compensatory polycythaemia).

It is for this reason that athletes wishing to increasethe O2-carrying capacity of their blood often train ataltitude.

Fig. 23 Production of blood cells.

Box 4 Clinical note: Use and abuse

of erythropoietin

One of the problems associated with chronic renalfailure is anaemia. This can be treated with injectionsof erythropoietin which substitute for normal renalproduction of the hormone, stimulating marrowproduction of red cells. Synthetic erythropoietin (EPO)

is also used by athletes, however, in order to increasehaemoglobin levels and improve performance withouthaving to train at altitude. This practice can lead todangerously high haematocrits, with risk of bloodclotting in arteries due to slow blood flow.

Erythrocytes


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Nutritional requirements for redcell production

Erythropoiesis and haemoglobin synthesis requireadequate supplies of the vitamins B12 (cyanocobala-min) andfolic acid, as well as the mineral iron. Defi-ciencies of these may cause anaemia.

Vitamin B12 and folateIf B12 or folate levels are reduced, cell division andmaturation are adversely affected. This is particu-larly important at sites of rapid cell turnover, suchas the bone marrow. There is a reduction in the redcell count so that the overall haemoglobin concentra-tion falls. The erythrocytes which do form areabnormally large (macrocytes), so this is known as amacrocytic anaemia. Abnormal erythrocyte precursorscalled megaloblasts are found in the marrow, so theterm megaloblastic anaemia is also used. It should be

appreciated that there is no problem with haemo-globin synthesis within the developing cells; thereare just too few red cells produced.

Deficiency of B12 or folate can arise in two ways.The diet itself may include inadequate amounts ofthe normal source foods for these vitamins, e.g.,animal products (for both B12 and folate) and greenvegetables (rich in folate). Vitamin B12 deficiency canalso occur with a normal diet as a result of reducedB12 absorption. Parietal cells in the stomach normallysecrete intrinsic factor, which binds to B12, and it isthe resulting complex that is absorbed from the

ileum (Section 6.4). The B12 is then transported to theliver, which normally stores 12 years supply (liveris an excellent dietary source of B12). In perniciousanaemia, there is reduced secretion of intrinsic factor.This leads to malabsorption of B12 and a megalo-

blastic, macrocytic anaemia results. These patientsoften also appear mildly jaundiced because ofincreased bilirubin production following haemo-lysis of the abnormally fragile red cells. Treatmentinvolves regular intramuscular injections with B12,thereby bypassing the normal intestinal absorptionmechanisms.

Iron

If the supply of iron is inadequate, haemoglobin syn-thesis is restricted. This leads to an anaemia in whicherythrocytes contain less haemoglobin than normal(they are hypochromic) and are, as a result, smallerthan normal (microcytic). Iron-deficiency anaemiacan occur whenever iron demand exceeds supply.That may be because of reduced iron intake orincreased iron loss, e.g., because of chronic bleeding.Men normally lose approximately 1 mg of iron daily,

chiefly through the shedding of intestinal epithe-lium. In menstruating women, this can rise to 2 mgor more. Since the average diet provides 1015 mgof iron each day, of which about 10% is normallyabsorbed, iron balance is usually maintained in men,

but women run an appreciable risk of becoming irondepleted. Pregnancy increases iron demand further

and iron supplements may be necessary.Iron is mainly absorbed in the ferrous form by

means of intestinal transferrin and an irontransfer-rin receptor (Section 6.4). The rate of absorptionincreases when iron demand is increased within the

body. Plasma transferrin transports iron withinthe circulation and excess iron is stored asferritin orhaemosiderin, particularly in the liver, spleen and

bone marrow. Mean iron stores are 23 times largerin men than women, presumably as a consequenceof menstrual losses.

Erythrocyte sedimentation rateIn an undisturbed vertical column of anticoagulated

blood, erythrocytes slowly settle out, leaving a clearcolumn of plasma above them. This rate of sedimen-tation increases in certain disease states and theerythrocyte sedimentation rate (ESR) is a widelyused clinical investigation. Normal values lie in therange 510 mm h1 but may be higher during preg-nancy. Abnormally high ESR values are often associ-ated with an increase in immunoglobulins. Thisfavours aggregation of red cells into stacks calledrouleaux, which sediment more rapidly than singlecells.

Blood groups and transfusion

The ability to replace blood lost following trauma orsurgery is a vital aspect of modern medicine. Thisrelies on an understanding of immune reactionsagainst red blood cells, since these may be fatal andmust be avoided if blood from one person (the donor)is to be safely transfused into another (the recipient).Whenever antigens on the surface of erythrocytes(agglutinogens) come into contact with specific anti-

bodies directed against them (agglutinins), the cellsclump together or agglutinate. By testing for aggluti-nation of red cells with known antibodies, the eryth-rocyte antigens can be identified and this defines the

blood group. A variety of different antigen typeshave been identified but those of the ABO and Rhesussystems are the most common.

ABO system

There are two main antigens in this system, A andB, and these give rise to four different blood groups


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as shown in Table 2. Of these, blood groups Oand A are almost equally common and togetheraccount for over 85% of the population in WesternEurope. It should be noted that plasma alwayscontains preformed antibodies (IgM class) againstA or B antigens which are not already presenton our own erythrocytes, whether we have beensensitized by exposure to foreign red cells ornot. This breaks the general rule in immune responses,since antibodies against all other foreign antigensare only secreted in appreciable amounts afterexposure to that antigen (Section 2.5). It may bethat we are all exposed to A and B antigens fromanother source, e.g., intestinal bacteria, and only

become tolerant to the antigens also present on our

erythrocytes. Whatever the mechanism, the conse-quence is that a major immune reaction can beexpected on the first exposure to blood of the wrongABO group.

Rhesus (Rh) factorBlood is either Rh positive or Rh negative dependingon whether red cells carry one of the Rh antigens ornot. There are three main Rh antigens, C, D and E,

but D is the most common. Inheritance is dominantso that the genotypes Dd and DD both result in D

positive blood. Over 85% of Western Europeans areRh positive.A Rh-negative recipient will mount an immune

response against Rh-positive blood but, unlike ABOagglutinins, there are normally no anti-Rh anti-

bodies in plasma from a Rh-negative individual.Therefore, there is unlikely to be any Rh-dependentagglutination following an initial transfusion withRh-positive blood. This exposure sensitizes theimmune system to the Rh antigen, however, so thatsubsequent mismatched transfusions can lead to

prompt agglutination and haemolysis of the donorcells. Rhesus sensitization can also occur when a Rh-negative mother gives birth to a Rh-positive baby.Fetal red cells, normally separated from the maternalcirculation by the placenta, may enter the mothers

blood during delivery as the placenta is sheared offthe uterine wall. This stimulates production of anti-

Rh antibodies by the mother and, if there is asubsequent Rh-positive pregnancy, these antibodies(IgG class) cross into the fetal circulation, leadingto haemolysis. The resulting jaundice, anaemiaand heart failure may threaten the babys life.These problems of the Rhesus baby have largely

been eradicated by injecting all D-negative motherswith anti-D antibodies shortly after the birth ofeach baby. Any circulating D-positive cells becomecoated with exogenous antibody and are destroyed

before they can stimulate the maternal immunesystem. This is an example of temporary passive

immunity (conferred by the injected immunoglobu-lins) preventing the development of permanentactive immunity. It should be noted that fetal/mater-nal ABO incompatibility is common but has no dam-aging consequences because the IgM class anti-Aand anti-B antibodies are too large to cross theplacenta.

Transfusions and cross matchingThe terms universal donor (blood group O, Rhnegative) and universal recipient (blood group AB, Rhpositive) are sometimes used to indicate conditionsin which transfusions may be attempted withoutknowing the blood group of both donor andrecipient. The reasoning is that the red cells of theuniversal donor carry no antigens and so cannot

be agglutinated, while the plasma of the universalrecipient contains no antibodies and could notagglutinate donor cells, regardless of their group.Possible agglutination of recipient cells is regardedas unlikely, since agglutinins in the donor plasmawill be diluted following transfusion. Use of O,Rh negative blood for a patient of unknown blood

group is reserved for life-threatening emergencies,however, and donor and recipient blood groupsshould normally match. Indeed, the existence of awide range of rarer erythrocyte antigens means that

blood of the appropriate group should actually betested against samples of the patients cells andplasma before being transfused. Donor cells aremixed with recipient plasma, while recipient cellsare mixed with donor plasma; there should be noagglutination in either case. This is referred to ascross matching.

Erythrocytes

Table 2 Summary of the ABO blood group system.Inheritance of the A and B antigens is autosomalcodominant so there are four possible combinations of redcell antigens. The plasma contains antibodies to anyantigen not present on the red cells

Genotype Antigens Blood Antibodieson cells group in plasma

OO None O Anti-A and anti-B

OA or AA A A Anti-B

OB or BB B B Anti-A

AB A and B AB None


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2.4 Leucocytes

body (Section 2.5) and can be subdivided into B andT lymphocytes.

Monocytes constitute 25% of leucocytes. Theyhave the greatest phagocytic potential of all bodycells. Tissue macrophages are believed to be mono-cytes which have migrated into the connectivetissues. The monocyte/macrophage system formsthe core of the reticuloendothelial system.

Reticuloendothelial systemThis phagocytic system is almost synonymous withthe tissue macrophage system. Mobile macrophagesrove freely through connective tissues, but manymore remain relatively fixed in a given region.When activated, however, these will also becomemobile, being chemotactically attracted to local sitesof infection or damage. There are dense aggregationsof macrophage-type cells within the reticular tissueof the lymph nodes, spleen and bone marrow. Alve-olar macrophages in the lung and the phagocyticKpffer cells in the liver, along with microglialcells in nervous tissue, are also regarded as part ofthe reticuloendothelial system, as are the bloodmonocytes.

Leucocyte production

Leucocytes originate from the pluripotential stemcells in the bone marrow, which divide and maturegiving two separate leucocyte stem cell lines(Fig. 23).

The myeloid line. This gives rise to the three typesof granulocyte as well as monocytes and macro-phages. These cells all have important phagocyticroles. The myeloid stem cell line also produces largemultinucleate megakaryocytes from whichplatelets arederived.

Learning objectives

At the end of this section you should be able to: state normal values for white cell count and

percentages of each leucocyte type (the

differential white cell count) explain how leucocytes are classified

morphologically and outline the function of eachtype

explain the term reticuloendothelial system

outline how leucocytes are manufactured.

White blood cells, or leucocytes, are vitally impor-tant in the disposal of damaged and ageing tissueand in the immune responses which protect usfrom infections and cancer cell proliferation.The total blood white cell count is normally in therange 4 109 to 10 109 L1 (410 103 mm3), but thismay increase markedly during infection orinflammation.

Leucocyte types

Based on their histological appearances, five maintypes of leucocyte may be identified. These fall intotwo morphological groups. Polymorphonuclear granu-locytes have irregular, multilobed nuclei and a highdensity of cytoplasmic granules (Fig. 23). Neutro-phils, eosinophils and basophils all belong in this

group. Lymphocytes and monocytes, by compari-son, lack granules and have large, regular nuclei,and so they are classified as mononuclear agranulo-cytes. The basic characteristics of individual leuco-cyte types are listed below.

Neutrophils comprise 6070% of circulatingleucocytes. They are highly mobile and can engulfdebris or foreign organisms through the process ofphagocytosis, trapping the target in a vesicle whichthen fuses with a lysosome (Fig. 24). Organic mate-rial is digested by lysosomal enzymes, althoughinorganic material may remain within the cytoplasm

indefinitely.Eosinophils make up 14% of circulating leuco-

cytes. They are phagocytic and are particularlyinvolved in the destruction of parasitic worms butmay also contribute to allergic responses.

Basophils generally account for under 0.5% ofleucocytes. These phagocytes release histamine andheparin and are involved in allergic responses.

Lymphocytes are the only nonphagocytic whitecells and represent 2530% of blood leucocytes. Theyare central to specific immune defences within the

Fig. 24 Phagocytosis of a bacterium.


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The lymphoid line. From this stem cell line thelymphocytes are produced (Fig. 23). B cells maturein the marrow before being distributed to thelymphoid tissues of the body, i.e. the lymphnodes, spleen, thymus and Peyers patches in theintestinal submucosa. T lymphocyte precursorcells are believed to migrate initially to the thymus,

where they mature fully before being redistributedto other lymphoid sites. Lymphocytes can replicateand develop further within the lymphoid tissuesof the body and there is a continuous recirculationof lymphocytes from blood to lymph and backagain.

2.5 Immune responses

Mechanical and chemical barriersagainst infectionThe epithelia covering skin and lining the gastroin-testinal, genitourinary and respiratory tracts providemechanical barriers which help exclude damagingorganisms. This is aided by mucus secretion in therespiratory tract, which traps dirt and pathogens

and allows them to be swept out by the action ofepithelial cilia. Chemical secretions, such as acid inthe stomach and vagina, also play a role and maydefend against colonization of the epithelial surface,e.g., preventing vaginal yeast infections.

The inflammatory response andphagocytosisInflammation is a set of local cellular and vascularresponses to tissue damage or infection which accel-erates the destruction and phagocytic removal of

invading organisms and debris.Phagocytic responses in inflammation Tissue mac-rophages adjacent to a site of bacterial invasion, forexample, become mobile and phagocytically active.Chemicals released from injured and infected cells(chemotaxins) act as attractants for these cells, direct-ing their movements towards the damaged area in aprocess called chemotaxis. Local macrophages aresoon reinforced by the migration of blood neutro-phils and monocytes into the region (Fig. 25). Thesestick to the endothelium of capillaries in the affectedarea (leucocyte margination) and then invaginate them-

selves through the clefts between the endothelialcells using active amoeboid movements (diapedesis).

Immune responses

Learning objectives

At the end of this section you should be able to: list and classify immune mechanisms asnonspecific or specific (innate or acquired)

describe the mechanisms of nonspecific (innate)immunity, paying particular attention to theinflammatory response and complement functions

describe the mechanisms of specific (acquired)immunity, differentiating clearly between antibody-mediated and cell-mediated responses

explain with examples the differences betweenactive and passive immunity.

The immune defences of the body are classically sub-divided into those which are nonspecific and innate,and those which are specific and acquired. This is auseful division, but it should be remembered thatthere are many points of interaction between the twosystems. For example, lymphocytes only producespecific antibodies against foreign molecules whenthese antigens are first processed by nonspecificphagocytic cells such as macrophages. At the sametime, antibodies lead to antigen removal by am-plifying pre-existing, nonspecific responses. Thesetwo elements of immunity are, therefore, highlyinterdependent.

Nonspecific or innate immunity

This depends on interrelated defence mechanismswhich act against any foreign or abnormal cell, i.e.,they are nonspecific. They are also said to be innatesince they do not depend on previous exposure to aparticular organism. Nonspecific immune mecha-nisms include physical barriers to infection, inflam-mation, complement activation and natural killercell activity.

Fig. 25 Diapedesis of blood granulocytes and monocytes andchemotaxis of local macrophages increase tissue phagocyte densityduring inflammation.


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The blood-borne phagocytes escape into the tissueswhere they are chemotactically attracted to assist inremoval of infectious or toxic agents and tissuedebris. The resulting congregation of large numbersof neutrophils and macrophages within a tissue is thehistological hallmark of acute inflammation.

Vascular responses in inflammation Leucocyte

aggregation in inflamed tissues is accelerated byincreased blood flow caused by dilatation of local

blood vessels. This transports more white cells intothe region, while an increase in capillary permeabil-ity promotes diapedesis. These vascular changes arestimulated by the generation of a variety of vasodila-tor substances within damaged tissues.

Kinins. Activation of a cascade of proteolyticenzymes known as the kininkallikrein system pro-duces vasoactive kinins, particularly bradykinin.

Vasodilators. Basophils and mast cells release thevasodilators bradykinin, 5-hydroxytryptamine (sero-

tonin) and histamine, as well as the anticoagulantheparin.

Prostaglandins. Activated phagocytic cells alsostimulate prostaglandin synthesis and this mayamplify the mechanisms of inflammation. Drugswhich inhibit prostaglandin production, such asaspirin and glucocorticoid steroid hormones, can beused as antiinflammatory agents.

Local and systemic effects of inflammation Theinflammatory response leads to a number of charac-teristic effects at the site of injury or infection:

Increased blood flow causes redness and raisesthe temperature locally.

Increased capillary permeability allows fluid toleak into the tissues, causing local swelling(oedema). Plasma proteins, including clottingfactors, also leak out so that a meshwork offibrin clot is laid down in the interstitium. Thisprovides a mechanical barrier to the spread ofinfection.

The release of tissue-damage products stimulatesnociceptors, causing pain (Section 7.4).

Acute inflammation (particularly if caused byinfection) may also be associated with generalized(systemic) responses.

Body temperature. This may rise as a result ofresetting of the hypothalamic set point (Section 1.1).Fever (pyrexia) seems to increase the activity ofphagocytic cells and is promoted both by exogenouspyrogens, including bacterial products known asendotoxins, and by endogenous pyrogens released

by phagocytes. Endogenous pyrogens stimulateprostaglandin synthesis and this is blocked by drugs

such as aspirin. This probably explains their abilityto reduce fever (their antipyretic action).

Blood white cell count. There is an increase in the blood white cell count (a leucocytosis), most of theincrease being the result of extra neutrophils (a neu-trophilia). This reflects both the rapid mobilization ofneutrophils already present in the bone marrow and

an increased rate of production in the marrow.

Activation of the complement systemForeign cells, especially certain bacteria, carry surfacemolecules which activate the complement system ofplasma proteins. These normally exist as a family ofinactive precursors which are activated by proteo-lytic cleavage. The system is organized as a cascadeso that each activated component activates the nextin the sequence (Fig. 26). Thus, activating one com-ponent can lead to activation of the entire comple-ment system. Nonspecific activation is referred to as

activation by the alternate pathway, distinguishingit from the classical pathway to activation, whichrequires antibody.

Complement function Complement activation defendsagainst infection both by killing cells directly and

by promoting phagocytosis (Fig. 26).Direct cell killing. The activated forms of the

last five components in the system (C5C9) com- bine to form a protein called the membrane attackcomplex. This becomes inserted in the plasma mem-

brane of the invading cell, where it forms a largepore. When the density of such pores is high, cell

lysis results.Phagocytosis. Complement activation produces a

series of biologically active complement fragmentswhich increase the efficiency of phagocytosis. Thisinvolves several mechanisms:

Opsonization makes target cells more susceptibleto phagocytosis. Complement fragments, whichact as opsonins, bind to the surface of bacteria.Phagocytic cells, which carry receptors for thesefragments, become attached to them and thisincreases the efficiency of bacterial phagocytosis.

Chemotaxis by complement fragments attractsmore phagocytic cells to an infected or damagedregion.

Vasodilatation occurs and capillary permeabilityis increased following complement activation,thus amplifying the inflammatory response.

Natural killer cellsThese are lymphocyte-like cells with granular cyto-plasm. They can identify and destroy tumour cellsor cells infected with viruses. The mechanism is not


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specific to any particular virus or tumour type and

previous antigen exposure is not necessary.

Nonspecific regulators of immunityThere are a large number of molecules, other thanspecific antibodies, which are important in regulat-ing immune defences. Interferons are proteins whichcan be released by any virus-infected cell and theyinhibit viral replication in other cells. Activated leu-cocytes and macrophages also produce a variety ofnonantibody proteins which participate in the regu-lation of immunity. Phagocytic cells (granulocytes,monocytes and macrophages) secrete cytokines, whilelymphocytes produce lymphokines. These lympho-kines can amplify nonspecific immune mechanismswhenever a lymphocyte is activated by an antigen.Any product of an immune cell (other than an anti-

body) which signals to other immune cells may alsobe termed an interleukin.

Specific or acquired immunity

Specific immunity refers to a number of mechanismswhereby susceptibility to infection by a particular

organism is greatly reduced following initial expo-sure to it. This is clearly demonstrated with measles,mumps, rubella and other childhood infections, inwhich the immunity acquired following a first infec-tion usually protects us if we are exposed to the sameorganism later in life. Indeed, clinical infection isnot necessary; vaccination uses weakened or killed

pathogens to stimulate immunity without produc-ing illness. In both infection and vaccination the pro-tection acquired is highly specific; having had a boutof measles does not reduce ones chances of contract-ing rubella or mumps, for example.

Specific immunity relies on the ability of theimmune system to respond to foreign molecules,which are called antigens, and the defence mecha-nisms thus activated are targeted against the relevantantigen. Antigens are usually large (molecularweight >10 000), complex molecules. Proteins areparticularly antigenic, although polysaccharides,

lipoproteins and glycolipids can also act as antigens.Much smaller molecules, known as haptens, may alsostimulate an immune response, but only if attachedto a protein for initial presentation to the immunesystem. Following sensitization, however, subse-quent responses can be stimulated by the haptenmolecule alone. This mechanism accounts for aller-gic reactions to small organic molecules, includingantibiotics like penicillin, and even to inorganicspecies such as nickel.

Specific immune responses rely on lymphocytesand may be mediated either by antibodies (humoral

responses) or cells. Antibody-mediated immunitydepends on B lymphocytes while T lymphocytes areresponsible for cell-mediated immunity.

Antibody-mediated responsesThese are particularly important in protection against

bacterial infections, although they play a role inimmunity against some viruses.

B lymphocyte activation This is the first step in anti-body production and it occurs when foreign antigens

come into contact with B lymphocytes. These cellscarry surface immunoglobulins, or antibodies, whichact as antigen-specific membrane receptors. Whenthese bind to the relevant antigen, the antigen-receptor complex is endocytosed by the lymphocyte,which is then activated. Each B cell will only bindwith one type of antigen and this leads to productionof circulating antibodies with the same specificity. Itis important to appreciate that although B lympho-cytes specific for all possible antigens appear to bepresent in the body, they do not normally secrete

Immune responses

Fig. 26 Nonspecific activation of the complement cascade by thealternate pathway.


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antibody. Specific antibody only appears in bodyfluids once the relevant B cell has been activated byantigen binding, after which it multiplies and thedaughter cells become transformed intoplasma cellsand memory cells (Fig. 27). Plasma cells act as facto-ries for the relevant antibody, which they secrete inlarge amounts. Memory cells are identical with the

parent lymphocyte and they greatly expand thereserve of cells capable of being activated duringsubsequent antigen exposure. Activated B lympho-cytes also carry processed antigen in association withnormal surface antigens known as histocompatibilityantigens on their cell membrane. This allows them toact as antigen presenting cells which can in turnactivate T lymphocytes (see below).

Primary and secondary responses On first exposureto an antigen, the immune response, as indicated

by the plasma antibody concentration (or antibodytitre), takes some weeks to develop fully (Fig. 28).

This is referred to as the primary response and,although it can limit the duration of an infection, itis generally too slow to prevent it altogether. Anti-

body levels fall again as time passes, but a secondexposure to the same antigen produces a much morerapid antibody response which peaks at a higherconcentration and declines more slowly. Thissecondary response depends on the memory cellsformed following initial B lymphocyte activation.These provide a reservoir of lymphocytes which can

be rapidly activated during future exposure to therelevant antigen. This amplified antibody response

may overwhelm a potential pathogen before it cancause the symptoms of infection, i.e., it providesacquired, antibody-mediated immunity.

Antibody structure Antibodies belong to the class ofproteins known as gamma globulins or immunoglob-ulins (Ig). The basic monomer unit consists of twoheavy and two light peptide chains linked together

by disulphide bridges to produce a Y-shaped struc-ture (Fig. 29). Each monomer has two antigen-

binding sites, the specificity of which is determined by their amino acid sequence. This region is oftenreferred to as the variable region of the antibody,since the seemingly limitless range of antigen speci-ficities is produced by equally numerous variationsin amino acid sequence. The make-up of the rest ofthe antibody, however, does not change with chang-ing antigen specificity. Differences in the structureof this constant region allow immunoglobulins to

be divided into five different antibody classes (see

below). Enzyme digestion can split antibodies intotwo fragments, an antigen-binding fragment (Fab)and a crystallizable fragment (Fc) (Fig. 29). Varia-tions in the Fc region account for the differentproperties of different antibody types:

IgG is the most abundant Ig in blood and isproduced in large quantities during thesecondary response. It promotes phagocytosisand cell lysis.

IgM molecules are pentamers constructed fromfive basic monomer units (Fig. 29B) and are

secreted early on in antibody responses. IgMpromotes agglutination, phagocytosis and celllysis.

IgD is a surface antibody on B lymphocytes butits role is unclear.

Fig. 27 B lymphocyte activation by antigen binding leads toformation of antibody-secreting plasma cells and antigen-sensitivememory cells.

Fig. 28 Plasma antibody levels following first and secondexposures to a given antigen.


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IgA molecules are dimers formed from two

monomer units (Fig. 29C). IgA is present insecretions including saliva, tears and breastmilk.

IgE is important in parasitic infections and someallergic responses. It stimulates release of

bradykinin, histamine, 5-hydroxytryptamine andother inflammatory mediators from mast cellsand basophils.

Antibody function Antibodies bind to antigens, thuspromoting their destruction through a range ofdifferent mechanisms:

1. Targeting and amplification of nonspecificimmunity is the major strategy employed byantibody-mediated immunity to provide protec-tion against specific pathogens. IgG and IgMclass antibodies are particularly important inpromoting bacterial lysis and phagocytosis inthis way (Fig. 30A). Antibodies activate the complement system

by the classical pathway. The Fc region ofantigen-bound antibodies can bind and cleavethe C1 complement component. This leads to

activation of the entire complement cascadewith the effects described above. Antibodies can act as opsonins, using

receptors for the Fc region to bind phagocytesto antigens.

Antibody-coated cells are more likely to beattacked by natural killer cells.

2. Agglutination refers to the clumping together ofbacteria or foreign cells into large-scale latticesheld together by antibody linkages (Fig. 30B).This is possible because all antibodies have

more than one antigen-binding site and so can

interconnect target cells, physically hinderingthe spread of infectious agents and increasingthe likelihood of phagocytosis. IgM antibodiesare the most effective because their pentamericstructure has 10 binding sites per molecule (Fig.29). Agglutination reactions are used in bloodgroup testing (Section 2.3).

3. Neutralization of toxins and inactivation ofsome viruses may result from immunoglobulinscloaking biologically active sites (Fig. 30C). Thisis how antisera to snake and insect bites work.

Cell-mediated immunityThis relies on T lymphocytes, which do not manu-facture circulating antibodies. It is particularlyimportant in combating viral and fungal infectionsas well as in immune responses against potentialcancer cells.

T lymphocyte activation T cells are activated byexposure to a foreign antigen which is identified and

bound by specific surface receptors. This recognitionstep only occurs, however, if the antigen is closelyassociated with other, normal, cell surface antigens,

known as histocompatibility antigens (Fig. 31). Thiscan come about in one of two main ways:

1. Cytotoxic T lymphocytes can be activateddirectly by body cells carrying abnormal anti-gens on their surface. This occurs during viralillnesses, since foreign viral antigens areexpressed on the plasma membrane of anyinfected cells. Cancer cells also carry abnormalsurface antigens, which can lead to T lym-phocyte activation, and this may prevent the

Immune responses

Fig. 29 Immunoglobulin structure.

A

B C

Fig. 30 Mechanisms of antibody (Ab) function. (A) Targeting ofnonspecific immunity. (B) Agglutination reactions. (C) Neutralizationof toxins.


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development of clinical disease. Cell-mediatedimmunity is also important in organ transplantrejection when the donor antigens do not exactlymatch those of the recipient. As their nameimplies, cytotoxic T cells can kill the abnormalcells directly.

2. Helper T lymphocytes can be activated byexogenous antigens, such as those derived from

bacteria and environmental allergens. Theseantigens must first be processed by antigenpresenting cells (sometimes referred to asdendritic cells), however, which engulf theforeign agent and then break it down usinglysosomal enzymes. Individual antigens canthen be presented on the surface of the antigenpresenting cell in association with the normalhistocompatibility antigens those cells carry.Macrophages and B lymphocytes both act asantigen presenting cells.

T lymphocyte function As with B lymphocytes, Tcell activation leads to multiplication and matura-tion. There are three main types of T lymphocyte,however, each with a different function.

Cytotoxic T lymphocytes can lyse cells carryingthe antigen to which they are sensitive and areparticularly important in the immune response toviruses and cancer cells.

Helper T lymphocytes are vital in both antibody-and cell-mediated immunity, but they have no directeffects on foreign antigens or cells. They are acti-vated by antigen presenting cells such as macro-phages or activated B lymphocytes, rather than cellsurface antigens on infected or mutated bodycells as is the case for other T lymphocytes. Once

stimulated by the appropriate antigen, they releasea number of lymphokines, which stimulate otherimmune cells. This increases macrophage activity aswell as promoting multiplication and activation of Tand B lymphocytes. Helper T lymphocytes are thusvital to all immune responses.

Suppressor T lymphocytes inhibit lymphocyte

function. This may provide a mechanism for thecontrol of the immune response which reduces therisk of immune damage to normal body cells.

Cell-mediated responses persist longer thanantibody responses since activated T lymphocytessurvive longer than plasma cells. T lymphocyteimmunity also demonstrates memory, with morerapid and vigorous responses to a given antigen onrepeated exposure.

Active and passive immunity

Activation of lymphocytes provides active defence

against infection. If antibodies are injected or absorbedinto the body these will also protect us from a givenantigen. This is called passive immunity but, unlikeactive immunity, it only provides short term protec-tion (a few months) since there is no production ofmemory cells. A physiological example of passiveimmunity is the protection a newborn baby gainsfrom the maternal immunoglobulins in its body (IgGcrosses the placenta and IgA is present in breastmilk). As these antibodies are degraded over the first23 months of life, the level of passive protectionwanes, but by this time the infants immune system

is mature enough to mount active responses. In adultlife, injections of immunoglobulin may be used togive short term passive protection, e.g., against someforms of viral hepatitis.

Histocompatibilityantigen

Bindshistocompatibility

antigen

Fig. 31 T lymphocyte activation by cell surface antigen. Bindingrequires the presence of both foreign and normal histocompatibilityantigens. The activating cell may be any body cell carrying anabnormal antigen or a specialized antigen presenting cell which hasphagocytosed antigenic material.

Box 5 Clinical note: Immunosuppression and

organ transplantation

One of the major barriers to successful organ trans-plantation is the difficulty in matching donor organantigens to those of the recipient. An exact match israrely possible because of the shortage of donororgans and the wide range of tissue antigens, and sothere is always a risk that an immune response will bemounted against the transplanted organ (graft rejec-tion). Immunosuppressant drugs are used to reducethis risk, but these inhibit immune responses in anonspecific fashion and leave the patient susceptibleto infections. Nevertheless, use of well-matched organsand careful management of drug doses can giveexcellent graft survival rates.


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2.6 Platelets and haemostasis

arachidonic acid derivative) released from theendothelium.

Clotting or coagulation

Platelet plugs are insecure, temporary structures butthey are quickly anchored in place by the formationof a fibrin clot. Clotting, or coagulation, depends ona family of plasma proteins known as clotting factors,

several of which are manufactured in the liver invitamin K-dependent reactions. They are normallypresent as inactive proenzymes but, once activated,each factor activates the next by proteolytic cleavage.This continues in a sequential fashion down thecoagulation cascade and finally results in theconversion of prothrombin (factor II) to activethrombin. Thrombin catalyses first the conversion offibrinogen (factor I) to fibrin monomers, and thentheir polymerization into cross-linked fibrin strands.The resulting clot, which traps erythrocytes andother blood cells, bridges the opening in the bloodvessel wall and secures the platelets in position.After some time, clot retraction pulls the damagededges of the blood vessel together, squeezing outclear serum (serum is the fluid left after plasma hasclotted).

Intrinsic and extrinsicclotting pathwaysClotting can be activated through two differentmechanisms, referred to as the intrinsic and extrinsicmechanisms or pathways (Fig. 33). Whenever bloodis exposed to an abnormal surface, such as collagenin the wall of a torn blood vessel, clotting occurs bythe intrinsic pathway. Aggregated platelets play animportant role, releasing a phospholipid cofactorcalled platelet factor 3. The extrinsic pathway is initi-ated when plasma is mixed with specific products oftissue damage, collectively known as tissue thrombo-plastin. This bypasses some of the initial steps inthe intrinsic pathway. The two pathways converge,however, at the activation of factor X, the step priorto thrombin formation. Several steps in both

Platelets and haemostasis

Learning objectives

At the end of this section you should be able to: state the normal platelet count

describe the mechanisms of haemostasis

explain how the clotting cascade works,differentiate intrinsic from extrinsic activation andname the factors involved in the last three(common) steps

outline the mechanisms of clot removal.

Platelets (or thrombocytes) are blood-borne cell frag-ments which contain organelles and enzymes but nonuclear material. They bud off from megakaryocytes,giant multinucleate bone marrow cells derived fromthe myeloid stem cell line (Fig. 23). The normal plate-let count in blood is 150 109 to 300 109 L1 (150300 103 mm3). The role of platelets is to help preventor stop bleeding, a process called haemostasis.Haemostasis involves vascular spasm, plateletplug formation and clotting (coagulation).

Vascular spasm

Damage to the wall of a blood vessel leads to rapidsmooth muscle contraction. This may help to narrowthe vascular defect and reduce blood flow. Vascularspasm, however, can only reduce or arrest blood lossin the short term, as the vessels eventually relaxagain. Other haemostatic mechanisms are requiredif bleeding is to be arrested permanently.

Platelet plugging

Platelets normally circulate freely, but when theyare exposed to tissue collagen following vasculardamage they become adherent, sticking to the vesselwall and to each other (Fig. 32). At the same time,they release a number of chemicals, including ade-nosine diphosphate (ADP) and a product of arachi-donic acid metabolism called thromboxane A2. Theseincrease platelet stickiness, resulting in positivefeedback favouring the formation of an ever largerplatelet plug, which can stop bleeding by occludingthe opening. The bleeding time (the time taken forvisible blood loss from a small puncture wound tostop) is usually less than 5 minutes, but this mayincrease considerably if platelet function is defective.Platelet adherence to undamaged blood vesselsis normally inhibited by prostacyclin (another

Fig. 32 Platelets form plugs in regions of vascular damage.


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pathways require Ca2+ which explains why Ca2+-chelating agents such as citrate are effectiveanticoagulants.

Fibrinolysis and clot removal

Blood clotting cannot be allowed to continue unin-hibited or blood vessels would soon become perma-

Fig. 33 Activation of the clotting cascade by the intrinsic orextrinsic pathways. Both pathways require Ca2+ and the intrinsic

pathway is accelerated by platelet factor 3 (PF3).

Box 6 Clinical note: Clotting defects

Deficiencies of clotting factors can lead to severebleeding problems. This may arise because of liverfailure or vitamin K deficiency, both of which inhibitsynthesis of several clotting factors. There may also bean inherited deficiency of nearly any clotting factor.The most common is haemophilia, the classic form of

which results from factor VIII deficiency caused byan X chromosome-linked genetic defect. Affectedindividuals are nearly always male, while the conditionis inherited through carrier females. Haemophiliacsbleed spontaneously from a variety of sites and thismay require treatment with concentrated extracts offactor VIII from normal plasma.

nently blocked. A number of natural substancesact as anticoagulants, inhibiting clot formationin normal vessels. There is also a mechanismfor removing clots after damage to a blood vessehas been repaired. This depends on the proteolyticenzyme plasmin, which is formed from the inactiveplasma protein plasminogen following activationof the coagulation cascade. Plasmin catalysesthe breakdown of fibrin, a process calledfibrinolysisand phagocytic cells then remove the clot debris.


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Multiple true/false questions

Each of the following statements consists of astem followed a number of possible endings. State

whether each statement is True or False. For eachstem, all, several or none of the statements may betrue.

1. Plasma:

a. proteins account for at least 70% of totalplasma osmolality

b. volume is proportional to 1/haematocrit atconstant blood volume

c. staining with haemoglobin may result fromhaemolysis

d. contains inactive hormone precursors e. proteins influence the erythrocyte

sedimentation rate

2. Erythrocyte formation:

a. takes place mainly in the marrow of longbones during adult life

b. may be stimulated by a reduction in arterialO2 content

c. may be reduced in chronic renal failure

d. normally produces new erythrocytes at a rate

closer to 2 1011

day1

than 2 1010

day1

in a70 kg adult male

e. may slow down following gastrectomy

3. ABO blood group status:

a. is defined by the antibodies in our plasma

b. is most commonly group AB in WesternEuropeans

c. may be different for a mother and her baby

d. is tested using erythrocyte coagulationreactions

e. is autosomally inherited

4. Circulating leucocytes:

a. are all derived from pluripotential stem cellsin bone marrow

b. chiefly consist of lymphocytes

c. may leave the circulation at sites ofinflammation

d. are derived from two separate committed celllines in bone marrow

e. known as monocytes form part of thereticuloendothelial system

5. Complement activation:

a. involves proteolytic cleavage of inactiveproenzymes

b. may be initiated through the Fc portion of anantibody

c. is an important element of both nonspecificand specific immunity

d. can occur by intrinsic and extrinsic pathways

e. promotes phagocytosis through formation ofthe membrane attack complex

6. B lymphocytes:

a. secrete circulating antibodies

b. carry antigen receptors on their surface

c. require helper T lymphocytes duringactivation

d. secrete cytokines

e. mature fully in bone marrow

7. Immunoglobulins:

a. of the IgM class have 10 antigen-binding sitesper molecule

b. of the IgA class can cross the placenta

c. of the IgE class can stimulate degranulationof mast cells and basophils

d. of the IgG class are dominant in secondaryantibody responses

e. consist of light and heavy peptide chainslinked by hydrogen bonds

f. of the IgM class act as anti-ABO agglutinins

8. Blood clotting:

a. requires Ca2+

b. is promoted by platelet plugging

c. defects usually prolong the bleeding time

d. is initiated by tissue thromboplastin in theintrinsic coagulation pathway

e. stimulates fibrinolysis

Single best answer questions

For each of the following questions choose the singlebest answer.

Questions

Self-assessment: questionsMasterMedicine


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52

Questions

1. A sample of blood is repeatedly ejected from asyringe through a small bore needle. Thehaematocrit for this sample is reduced from0.47 to 0.35 after this treatment. Whatpercentage of the red cells have beenmechanically destroyed during this procedure?

a. 12%

b. 45% c. 26%

d. 0.12%

e. 0.26%

2. At physiological pH, plasma proteins:

a. are anions

b. move towards the cathode duringelectrophoresis

c. are all globulins

d. are only found in the vascular space

e. cannot bind to Ca2+ ions

3. With regard to blood groups, a Rhesus vebaby:

a. must have had parents both of whom werealso Rhesus ve

b. will have anti-Rhesus antibodies in itscirculation at birth

c. may be affected in utero by the presenceof Rhesus antigen in the maternalcirculation

d. poses a risk to the well-being of the mother ifshe is Rhesus +ve

e. may be sensitized against the Rhesus antigenby exposure to Rhesus +ve blood

4. The presence of polymorphonucleargranulocytes in peripheral tissues:

a. results from specific, acquired immuneresponses

b. is promoted by local vasoconstriction

c. results in high tissue levels of antibody

d. is a hallmark of acute inflammation e. inhibits chemotaxis

5. T lymphocyte activation:

a. always depends on the function of antigenpresenting cells

b. always depends on the presence ofappropriate histocompatibility antigens

c. is only important in cell-mediated immunity,and not in antibody-mediated immunity

d. does not occur on first exposure to an antigen

e. leads to short term responses lasting daysrather than weeks

6. With regard to blood transfusion:

a. people of blood group AB, Rhesus +ve arereferred to as universal donors because they

carry no antibodies against relevant red cellantigens in their blood

b. people of blood group AB, Rhesus +ve arereferred to as universal donors because theycarry all the relevant blood group antigens ontheir red cells

c. people of blood group O, Rhesus ve arereferred to as universal recipients becausethey have neither blood group antigens norantibodies in their blood

d. people of blood group AB, Rhesus +ve are

referred to as universal recipients becausethey carry all the relevant blood groupantigens on their red cells

e. people of blood group AB, Rhesus +ve arereferred to as universal recipients becausethey carry no antibodies against relevant redcell antigens in their blood

Matching item questions

Theme: Leucocytes

OptionsA. Neutrophils

B. Eosinophils

C. Basophils

D. B lymphocytes

E. T lymphocytes

F. Monocytes

G. Plasma cells

H. Memory cells

I. Killer cells

For each of the descriptions below choose the mostappropriate option from the list above. Each optionmay be used once, more than once or not at all.

1. These cells promote the rapid secondaryimmune response.

2. These are highly phagocytic agranular cells.

3. These cells secrete antibody into thecirculation.


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Questions

4. These cells contribute to nonspecific immunityby destroying abnormal body cells.

5. These cells mature within the thymus.

6. These cells promote inflammation by releasingvasodilator substances.

Theme: Haemostasis

OptionsA. Platelets

B. Vascular spasm

C. Prostacyclin

D. Thromboxane A2

E. Ca2+

F. Factor X

G. Factor II

H. Fibrinogen

I. Plasmin

For each of the descriptions below choose the mostappropriate option from the list above. Each optionmay be used once, more than once or not at all.

1. An anti-thrombotic product of arachadonicacid metabolism.

2. Is chelated by EDTA, preventing coagulation.

3. Might be used as a clot-busting agent toremove thrombi within coronary arteries.

4. Is activated by thrombin in the coagulationcascade.

5. Promotes platelet plugging.

6. Is also known as prothrombin in its inactiveform.

Short notes

Write short notes on the following:

a. plasma composition

b. red cell destruction

c. phagocytic cells

d. chemotaxins

Modified essay

Questions

1. Which mechanisms normally prevent or limitbleeding and what stimuli activate them?

2. What role does positive feedback play inhaemostasis?

Many blood vessels are relatively fragile structures,and so there is a constant risk of blood loss

through damage to vascular tissue, even in theabsence of trauma.

Two simple tests of haemostatic function are the

bleeding time (time taken for bleeding to stop,normally 15 minutes) and the clotting time (time

taken for a blood sample to clot in a glass tube,normally 48 minutes).

Questions

3. Is the intrinsic or extrinsic coagulationmechanism activated when blood clots in a glasstube?

4. If the clotting time is prolonged regardless ofwhether the intrinsic or extrinsic mechanism is

activated, what does this suggest about the siteof the defect in the coagulation pathways?

5. If an individual has an abnormally longbleeding time but a normal clotting time, whatis the likely cause of the problem?

The risk of blood loss from the circulation is

paralleled by a risk of blockade of the bloodvessels by unwanted clots. When a clot develops

within an intact vessel, obstructing flow, this is

referred to as thrombosis. This is a major cause of

ischaemic heart disease and cerebrovascularproblems (e.g., strokes). Clots may detach fromtheir site of origin and circulate with the blood to

become lodged in some distant blood vessel, a

process calle em olism. Various mec anismselp prevent t ese unwante events.

Questions

6. What mechanisms help prevent blood clotsforming in normal vessels? What vascularabnormalities might promote thrombosis?

7. What mechanisms promote removal ofunwanted clot?

Data interpretation

The following results were obtained on analysis ofa blood sample from an anaemic patient:

Haemoglobin concentration: 8.3 g dl

Red cell count: 2.2 1012L1

Haematocrit (PCV): 0.25


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Questions

Questions

1. Comment on these results.

2. Use the values above (the primary indices) tocalculate the following values: mean cellvolume, mean cell haemoglobin, mean cellhaemoglobin concentration (the secondary

indices). Give the appropriate units in eachcase.

3. How would you classify this anaemia?

4. Suggest a deficiency state which may havecaused these abnormalities?

Clinical scenario

Questions

5. What is the patients ABO blood group? Whichof the ABO agglutinins (antibodies) would youexpect to find in his plasma?

6. Which observations in the agglutination tableact as positive controls (i.e., demonstrate that apositive result is obtained when expected) andwhich provide negative controls (i.e.,demonstrate that a positive result is notobtained when not expected)?

Viva questions

1. How is erythrocyte production controlled?

2. What different types of immune mechanismsdo you know?

3. What is meant by the term bleeding time?Why might the bleeding time be prolonged?

A young male adult is admitted to hospital. He

complains that he feels weak and tires easily. Onquestioning, it becomes clear that he has been

bleeding into his gastrointestinal tract, probablyfrom a gastric ulcer. The patient is also found to

have a mild fever (38.6C).

The medical house officer sends a sample of the

patients blood to the haematology laboratory in abottle containing a Ca2+-chelating agent. The

requested investigations include measurement of

he haemoglobin concentration and a blood cell

count. The results are given in Table 3.

Questions

1. What is the purpose of the Ca2+-chelating agentin the sample bottle?

2. How might the patients symptoms ofweakness and tiredness be explained on the

basis of the haematology results?

Table 3 Haematological analysis for a patients bloodsample

Variable Measured Normalvalue value

Haemoglobin 9.6 g dl1 1416 g dl1

Red cell count 3.3 1012 L1 46 1012 L1

Reticulocytes 9% 02%

White cell count 15.6 109 L1 410 109 L1

Platelet count 190 109 L1 150400 109 L1

It is decided that blood transfusion may be

necessary and so a further sample of blood is sentfor grouping and cross matching. The patients red

cells and plasma are separated by centrifugation

and the red cells tested for agglutination in the

presence of different agglutinins (antibodies)

known to be specific for the A or B antigens. The

results are s own in a le 4.

Table 4 Results from ABO blood group testing

Red cell Saline Anti-A Anti-B antibodygroup antibody

O

A + +

B + +

Unknown + + + + (patients cells)

+ +, agglutination

3. Comment on the reticulocyte count in the lightof the other blood results.

4. Which of the blood results is most consistentwith the patients fever and what may be thecause of both changes?


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Multiple true/false answers

1. a. False. Total osmolality is dictated bydissolved ions. Plasma proteins are

responsible for the colloid osmotic pressureat the capillary wall, however.

b. False. Plasma volume is proportional to[1 haematocrit] at constant bloodvolume.

c. True. Haemoglobin is released from lysederythrocytes.

d. True. For example, angiotensinogen.

e. True. By promoting rouleaux formation.

2. a. False. Although the marrow of long bones

contributes to erythropoiesis in children,membranous bones are almost exclusivelyresponsible in adults.

b. True. This stimulates renal erythropoietin.

c. True. Because of a lack of erythropoietin.

d. True. You can estimate this from othervalues in the text: blood volume = 70 ml kg1,so total blood volume = 4.9 L in 70 kgindividual; erythrocyte count = 5 1012L1,so total number of erythrocytes = 24.5 1012.Erythrocyte life span = 120 days, so replace-

ment needs=

24.5

10

12

/120=

2.0

10

11

cells day1.

e. True. Because of a lack of intrinsic factorand consequent vitamin B12 deficiency.Anaemia may take months to appear

because of liver stores of B12.

3. a. False. Defined by the antigens onerythrocytes. There is, however, a one-to-one correspondence between ABO bloodgroup and plasma antibodies; see Table 2.

b. False. AB is rarest; A and O are most

common. c. True. For example, a group O mother and

group A father may have a baby with eithergroup O or group A.

d. False. Agglutination reactions.

e. True.

4. a. True. As are erythrocytes and platelets.

b. False. Neutrophils normally account fortwo-thirds of all leucocytes.

c. True. This process is called diapedesis.

d. True. The myeloid and lymphoid lines(Fig. 23).

e.

True.

5. a. True. Each complement component isactivated in this way.

b. True. Important in antibody-mediatedimmunity.

c. True.

d. False. Classical and alternate pathways.

e. False. Membrane attack complex lyses cellsdirectly. It is complement fragments,cleaved during activation, which act asopsonins and enhance phagocytosis.

6. a. False. Plasma cells produced by activatedB lymphocytes secrete antibody.

b. True. These have the same antigen specific-ity as the antibody secreted after lympho-cyte activation.

c. True. B lymphocyte activation does not takeplace in the absence of helper T lymphocyteactivation.

d. False. Cytokines are secreted by granulo-cytes and monocytes; lymphocytes secrete

lymphokines. e. True. Unlike T lymphocytes which com-

plete their development in the thymus.

7. a. True. IgM has five monomer units permolecule with two binding sites permonomer.

b. False. Only IgG can cross the placenta; IgAis present in secretions and breast milk.

c. True. Releases vasoactive substances likebradykinin and histamine.

d. True.

e. False. The peptide chains are linked bydisulphide bridges.

f. True. The strong agglutinating action ofIgM reflects its multiple binding sitestructure.

8. a. True. In both intrinsic and extrinsicpathways.

b. True. Because of platelet release of aphospholipid called platelet factor 3.

Answers

Self-assessment: answersMasterMedicine


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56

Answers

c. False. Bleeding time is more dependent onplatelet function.

d. False. Tissue thromboplastin initiates theextrinsic pathway.

e. True. Through conversion of plasminogento plasmin.

Single best answers

1. c. 26%. This is calculated by expressingthe reduction in haematocrit as a % ofthe original haematocrit, since thehaematocrit gives us a measure of the redcell volume.

2. a. The negative charge promotes binding tocations such as H+ and Ca2+. Althoughcapillaries are relatively impermeable toproteins, some do diffuse into the interstitialfluid.

3. e. A Rhesus ve baby may have parents oneor both of whom may be heterozygous forthe relevant gene. Since the Rhesus gene isautosomal dominant, they will still beRhesus +ve. Rhesus ve individuals onlyproduce measurable levels of antibodyfollowing sensitization by exposure to theRhesus antigen.

4. d. The presence of a large number ofgranulocytes, usually mainly neutrophils, isa histopathological hallmark of acute

inflammation in that tissue. This ispromoted and targeted by specific immuneresponses but does not require them, as it ispart of the nonspecific, or innate immuneresponse. The movement of granulocytesinto the tissues is promoted byvasodilatation and by the presence ofchemotaxic agents in the damaged/infectedtissue.

5. b. The T lymphocytes can only recognizerelevant foreign antigens when they arepresented in the context of normalhistocompatibility antigens, either on thesurface of the damaged cell itself (cytotoxicT lymphocytes) or by an antigen presenting.Helper and suppressor T lymphocytes helpmodulate antibody-mediated, as well ascell-mediated responses.

6. e. AB, +ve is the universal recipient becausesuch individuals do not produce antibodiesagainst antigens A, B or the Rhesus antigen,

all of which they carry on their own redcells. Answer e. is favoured over d. as thepathological consequences of a mismatchedtransfusion result from the recipientsantibodies binding to the donor antigens,triggering a generalized immune responsein the donor.

Matching item answers

Theme: Leucocytes

1. H. The production of large numbers ofmemory cells following the first exposure toan antigen leads to a rapid and amplifiedresponse on subsequent exposure.

2. F. Monocytes form part of thereticuloendothelial system.

3. G. Plasma cells actively secrete antibody; B

lymphocytes, from which they are derived,do not.

4. I. Killer cells are nonspecific in the sense thatthey do not respond to any particularantigen, but destroy any cell with abnormalantigens.

5. E. The T in T lymphocyte stands for thymus.

6. C. Vasodilators released include bradykinin,histamine and 5-hydroxytryptamine.

Theme: Haemostasis

1. C.

2. E. This is the basis of some anticoagulantsused to prevent laboratory blood samplesfrom clotting.

3. I.

4. H. Fibrinogen is activated by thrombin (activefactor II) to form fibrin, the molecularskeleton of a clot.

5. D. This is another arachadonic acid metabolite.

6. G.

Short note answers

a. Plasma consists of an aqueous solution ofelectrolytes, proteins and nutrients (glucose,amino acids, lipids and fatty acids). Thedominant cation is Na+ (140 mmol L1), withsmaller concentrations of K+ (4.5 mmol L1)and Ca2+ (2.5 mmol L1). The main anions areCl (105 mmol L1) and HCO3

(25 mmol L1).


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Answers

Proteins include albumin (osmotically activeand transport functions), globulins (trans-port, signalling molecules and immuno-globulins) and fibrinogen (clotting).

b. Red cell destruction mainly occurs withinthe spleen and becomes more likely as a cellages, mean life span being 120 days. Haemo-

globin is degraded in reticuloendothelialcells, releasing iron and globin molecules,which are broken into amino acids forfuture protein synthesis. The porphyrinring forms bilirubin, which is excreted in

bile.

c. Phagocytic cells engulf foreign material anddebris and destroy them using lysosomalenzymes. They are mobile and can beattracted to sites of inflammation by chemo-taxic agents. Phagocytic cells includemonocytes and all three types of polymor-

phonuclear granulocyte (neutrophils,basophils and eosinophils) in blood, as wellas tissue macrophages, which are believedto be monocytes which have escaped fromthe circulation. There are also phagocyticKpffer cells in the liver and microglial cellsin the brain. These, along with monocytesand macrophages (especially in the lymphnodes, spleen and bone marrow), constitutethe phagocytic reticuloendothelial system.

d. Chemotaxins are chemicals which mobilizeand attract phagocytes. Important examples

include tissue damage products and com-plement fragments. In acute inflammation,these agents lead to the aggregation of alarge concentration of neutrophils andmacrophages in the damaged tissues as partof the nonspecific immune response.

Modified essay answers

1. The haemostatic mechanisms are vascularspasm (in response to damage to vascularsmooth muscle or the surrounding tissues),plugging of vascular openings by aggregatedplatelets (activated by exposure of platelets tocollagen) and clotting (activated by collagenexposure, or contact with tissuethromboplastin).

2. Positive feedback is particularly important inplatelet plugging. Adherent platelets releasethromboxane A2 and ADP, both of whichfurther enhance platelet stickiness thus

increasing the size of the platelet plug. Thereare also positive feedback elements withinthe coagulation process since some activatedclotting factors promote earlier steps in thecascade, as well as directly activating the nextfactor in the chain.

3. This is the intrinsic coagulation mechanism.

No extrinsic agent is added to the blood butcontact with the abnormal surface (glass)activates clotting.

4. This suggests a defect in those stages of thecoagulation cascade which are common to bothpathways, i.e., factor X, prothrombin orfibrinogen (Fig. 33). It could also indicate areduced plasma concentration of Ca2+, sinceCa2+ is necessary for both mechanisms.

5. This suggests an abnormality of plateletfunction, possibly caused by a reduced platelet

count. Bleeding time is normally shorter thanclotting time and is largely determined by therate of platelet plug formation.

6. Rapid blood flow is an important mechanicalfactor, making it difficult for platelets to attachto the endothelium. Normal endothelial cellsalso release prostacyclin, which reducesplatelet stickiness, and plasma contains anumber of anticoagulants, such asantithrombin III, which inhibit fibrinformation. Sluggish blood flow (particularlylikely in veins) and damage to the endothelium

favours platelet adhesion and local coagulationleading to clot formation.

7. Activation of the clotting cascade also leads toconversion of plasminogen to plasmin, afibrinolytic enzyme. Clot debris is removed byphagocytic cells.

Data interpretation answers

1. All three parameters are lower than normal.

2. Mean cell volume (MCV) = (haematocrit)/

(red cell count) = 0.25/(2.2 1012) L = 0.114 1012 L = 114 fl (1 fl = 1015 L).

3. Mean cell haemoglobin (MCH) = (haemoglobinconcentration)/(red cell count)

4. The haemoglobin concentration needsto be expressed L1, not dl1, i.e., it has to bescaled up by a factor of 10. Therefore,MCH = 83/(2.2 1012) g = 37.7 1012 g =37.7 pg (1 pg = 1012 g).


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Answers

5. Mean cell haemoglobin concentration (MCHC)= MCH/MCV = (haemoglobin concentration)/(haematocrit) = 8.3/0.25 g dl1= 33 g dl1.

6. This is a macrocytic anaemia, i.e., the red cellsare larger than normal (increased MCV). TheMCH is also increased, because of theincreased cell size, but the MCHC is not

altered. This suggests there is a problem withcell division within the bone marrow but thathaemoglobin production can occur as normal.

7. Macrocytic anaemia has a range of causes butdeficiencies of vitamins B12 and/or folate areamong the relatively common causes.Microcytic anaemia, on the other hand,typically results from iron deficiency.

Clinical scenario answers

1. It acts as an anticoagulant by removing thefree Ca2+ necessary for the coagulation cascade.

2. The patient is anaemic, i.e., he has a reducedconcentration of haemoglobin. Fatigue is acommon symptom of anaemia, presumably

because of the reduced O2-carrying capacity ofthe blood.

3. An elevated reticulocyte count suggests anincreased rate of erythrocyte production in themarrow. This is a response to the anaemia,mediated by renal release of erythropoietin.

4. An elevated white cell count and fever are

both systemic features of inflammation, oftencaused by bacterial or viral infection.

5. Blood group is AB. There should be no ABOantibodies in his plasma (otherwise they wouldagglutinate his own red cells).

6. All the tests using saline are negative controls.This excludes nonspecific agglutination. Thetests using known cells provide both positiveand negative controls. For example, group Ocells should not agglutinate with any of theantibodies (all negative controls), while

group A cells should agglutinate with anti-A(positive control) but not with anti-B (negativecontrol).

Viva answers

1. It is important to emphasize the role of renalerythropoietin as the physiological stimulus tored cell production in the marrow. You shouldmake it clear that erythropoietin is produced inresponse to the low tissue O2 levels which

result from anaemia rather than being a directresponse to low haemoglobin. This explainswhy haemoglobin levels can actually be raisedfollowing chronic arterial hypoxia (decreasedarterial O2 content), e.g., due to disease or highaltitude (Section 4.6). Causes of anaemia maywell be asked about and the discussion may goon to consider sites of red cell production atdifferent ages or the significance of areticulocytosis.

2. This is a huge topic with great scope forgetting lost. Start with some general principles

of classification, e.g., nonspecific and specificimmunity. Build on this by listing the mainmechanisms involved under each heading. It islogical to discuss nonspecific mechanisms first

because many specific mechanisms act totarget and amplify these. It is likely that theexaminer will direct you with some subsidiaryquestioning at this stage. You should expect to

be asked about inflammation, antibody-mediated immunity or cell-mediated immunityin more detail.

3. Bleeding time is defined as the time taken for a

small wound (usually inflicted with a lancetinto the ear lobe) to stop bleeding. You should

be able to state a normal value. It is importantto identify the three mechanisms ofhaemostasis (vascular spasm, platelet pluggingand coagulation) and then you shouldcomment that platelet defects/deficiencies arethe most likely to affect bleeding time,although any of the three may cause bleedingto be prolonged. Questioning is likely to go onto deal with factors affecting platelet stickiness

but the discussion is also likely to take in

activation of the coagulation cascade.

Blood and Related Physiology

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