The principle function of the two tests that are the focus of this chapter is detection and monitoring of inflammatory and infectious disease. Both tests serve essentially the same purpose. Erythrocyte sedimentation rate (ESR), the older and more ­established of the two, is performed in the haematology laboratory using a whole blood sample. Plasma C-reactive protein (CRP) is an alternative test to the ESR, usually performed in the immunology or clinical chemistry laboratory using a plasma or serum sample. The CRP test has some advantages over the ESR. The diagnostic information that the two tests provide can sometimes be complementary; under these circumstances it is useful to have the results of both tests. We begin with consideration of the ESR.

Erythrocyte sedimentation rate (ESR)

The ESR test is one of the oldest and simplest tests still performed in clinical ­laboratories, and is based on a very visible phenomenon, familiar to all those who have collected blood. If a blood sample, collected into a tube containing an ­anticoagulant, is left undisturbed then the red cells (erythrocytes) gradually fall or sediment to the bottom of the container, leaving the clear straw coloured, plasma fluid above. At the end of the nineteenth century, physicians investigated this ­phenomenon and discovered that the red cells in a blood sample taken from healthy volunteers sediment slowly, but that the cells in a blood sample taken from those suffering a range of disease sediment much faster. From these observations the ESR test was born.

Despite minor modifications, measurement of ESR has remained essentially unchanged since its introduction nearly a hundred years ago. A narrow bore tube of standard length is filled with anticoagulated blood and placed in a vertical ­position. The tube is left undisturbed for a defined time (usually one hour). During that time the erythrocytes sediment leaving an increasingly large column of clear plasma above. After one hour has elapsed the distance from the top of the tube to the interface between clear plasma and red cells is measured. This distance is the ESR expressed in millimetres per hour. In short the ESR is the distance in millimetres red cells fall in one hour.

Normal physiology: what affects red cell sedimentation?

Although apparently simple, the rate at which erythrocytes sediment is a complex phenomenon, which even now is not entirely understood. Clearly red cells fall due to gravity, because they have a greater density then the plasma in which they are suspended. Red cells have a net negative charge due to the presence of ­membrane bound proteins on their surface. This electrostatic force tends to make red cells repel each other. This is the situation in health; red cells are for the most part separate and fall individually. If for any reason this tendency of cells to repel each other is overcome, then they aggregate together to form ‘rouleaux’ (red cells stacked together rather like a pile of coins). Since an aggregation of red cells has greater density than single cells aggregated cells sediment faster. It is this ­abnormal tendency for cells to overcome their natural repulsion for each other, and ­aggregate, which explains the increased ESR found in disease. The crucial ­question is: what makes red cells aggregate? The answer lies in the plasma in which red cells are suspended. Certain proteins in plasma, most notably fibrinogen and immunoglobulins, act as molecular bridges between red cells. When present in high concentration the effect of these proteins is a marked increase in the ­aggregation of red cells. As will become clear, it is disease states that are ­associated with abnormally high concentration of these proteins in plasma which most ­commonly result in a raised ESR.

In addition to the composition of the plasma in which they are suspended the rate at which red cells sediment is also affected by both numbers and shape of red cells themselves. So that, for example, a significant decrease in the number of red cells as occurs in anaemia is associated with an increase in ESR whilst an abnormal increase in red cell numbers (polycythaemia) reduces ESR. The shape of red cells of those suffering sickle cell anaemia is abnormal; these so called sickle cells sediment slower than normal red cells.

Laboratory measurement

Patient preparation

No particular patient preparation is necessary.

Sample timing

Depending on the method being used, a delay of more than a few hours in ­processing samples can affect results. Samples stored overnight may be unsuitable for analysis. It is therefore best practice to take samples at a time that coincides with routine transport to the laboratory.

Sample requirements

A sample of venous blood is required. Most laboratories provide a specific tube for ESR only (black top) which contains the anticoagulant sodium citrate. The required volume is printed on the label. It is essential that anticoagulant in the bottle is mixed with the blood by gentle inversion.

Interpretation of results

Approximate reference range

Causes of a raised ESR

General considerations

ESR increases gradually with age, rising at the rate of around 0.8 mm/hr every five years. From the fourth month of pregnancy ESR usually rises to a peak of 40–50 mm/hr, returning to normal after birth.

ESR is one of the least specific of all laboratory tests. In other words, like a raised temperature or heart rate, a raised ESR occurs in many different sorts of illness. The changes in plasma proteins which give rise to increased red cell aggregation and raised ESR are a feature of any illness associated with significant tissue injury, inflammation, infection or malignancy. Unfortunately, from a diagnostic point of view, in most of these disease states, it is possible for the ESR to be normal. Furthermore it is clear that ESR is occasionally raised in normal healthy individuals. Despite these awkward anomalies that tend to confound interpretation of an ESR result, the ESR continues to be used in clinical practice. In general the higher the ESR the greater is the likelihood of a significant inflammatory, infectious or malignant disease.

Inflammatory disease

The inflammatory response to tissue injury results in abnormal increase in the synthesis of plasma proteins including fibrinogen, which tend to promote rouleaux formation and raise ESR. Potentially then any disease with an acute or chronic inflammatory component may be associated with an increase in ESR. In clinical practice the test is used as supportive evidence of inflammation in the diagnosis of disease associated with chronic inflammation such as rheumatoid arthritis, Crohn’s disease and ulcerative colitis. It is frequently used to monitor disease activity in these conditions. A rising ESR in a patient with a known chronic inflammatory condition, such as rheumatoid arthritis, implies that disease activity is continuing or increasing and therefore not responding to current therapy. Conversely a falling ESR indicates reduced inflammation and therefore response to therapy.

Although the ESR has a limited diagnostic role for most diseases with an inflammatory component, because there are other more reliable and specific tests available, there are two related conditions in which ESR is often the only laboratory test that is abnormal. These are temporal arteritis (sometimes called giant cell arteritis) and ­polymyalgia rheumatica. The first is an inflammatory disease of arteries, usually in the head and neck. The condition is relatively common in the elderly causing general feeling of malaise and tiredness along with severe headaches; sudden blindness may occur if the optic artery is affected. The second is an inflammatory condition affecting muscles causing severe muscle pain and stiffness particularly after resting. The two conditions often appear together in the same patient and both are associated with very high ESR, usually greater than 75 mm/hr, often higher. The ESR gradually returns to normal during treatment with steroids, and the test is used to monitor response to therapy. These are the only conditions in which ­diagnosis depends on the ESR test.

Infectious disease

Infection may be associated with an increased ESR. In general, bacterial infections tend to result in an increased ESR more frequently than those caused by viruses. Particularly high ESR (i.e. greater than 75 mm/hr) is most frequently found in those suffering chronic infections, for example, TB and sub-acute bacterial endocarditis (infection of valves of the heart), but any bacterial infection, if sufficiently severe, may be associated with very high ESR.

Malignant disease

Many patients suffering cancer of all types have a raised ESR. However, since a significant proportion of cancer patients do not have a raised ESR, the test has no place in cancer diagnosis. In the absence of infectious or inflammatory disease a significant increase in ESR (i.e. >75 mm/hr) might suggest that further investigation to detect cancer is warranted. Some authorities believe that a particularly raised ESR (i.e. >100 mm/hr) in a patient with cancer is reliable evidence of tumour spread beyond the primary site (metastasis).

The only widely accepted use of the ESR so far as malignant disease is concerned is in the diagnosis of multiple myeloma, a malignant disease of bone marrow in which uncontrolled proliferation of plasma cells within the bone marrow causes bone pain and bone destruction. These malignant plasma cells synthesise huge quantities of abnormal immunoglobulin at the expense of normal immunoglobulin (antibody) production. Since immunoglobulin is one of those proteins which increase rouleaux formation, and thereby the ESR, multiple myeloma is almost always associated with an increase in ESR (often >100 mm/hr). So consistent is this finding that a raised ESR was once among the criteria required for diagnosis of multiple myeloma.

Finally ESR is almost always raised in patients with Hodgkin’s disease (­malignant tumour of lymph nodes). The ESR is not used to make a diagnosis but is frequently use to monitor disease progress and therapeutic effectiveness.

Other common causes of raised ESR

Myocardial infarction (heart attack) involves tissue injury to heart muscle (­myocardium). The consequent inflammatory response to this injury includes increased synthesis of plasma proteins (fibrinogen), which causes increased red cell aggregation and therefore raised ESR. Thus myocardial infarction is a common cause of raised ESR. Typically ESR rises after an infarct, peaking one week later. A gradual return to normal is usual over the next few weeks. A raised ESR is also common in patients suffering renal disease.

Causes of reduced ESR

A reduced ESR is far less common than an increased ESR and actually of little clinical significance. An abnormally high red cell count (polycythaemia) is the most frequent cause. Rare causes include sickle cell anaemia and hereditary spherocytosis; both conditions are associated with abnormally shaped red cells which slow the rate at which they sediment.

C-reactive protein (CRP)

Normal physiology – synthesis and function

C-Reactive protein (CRP) is so called because the first of its properties to be identified at the time of its discovery, in 1930, was its ability to react with (precipitate) C-polysaccharide, a constituent of the wall of streptococcal bacteria. CRP is synthesised in the liver and is one of a number of proteins present in blood plasma that are collectively called the acute phase proteins. Fibrinogen, a protein already discussed for its significance in the ESR test, is another of these acute phase proteins. In health acute phase proteins are present in blood plasma at low concentration. However following any tissue injury, infection or acute inflammation, blood concentration rises. This acute phase reaction, as it is called, is one part of the body’s overall complex protective response to tissue injury or microbial attack. The ESR test owes its clinical utility to the acute phase reaction, because it is the increase in plasma concentration of the acute phase protein fibrinogen that accounts for the increased rate of red cell sedimentation associated with disease.

CRP is considered the archetypal acute phase protein. Under the influence of a chemical messenger called interleukin 6 (IL-6) released from macrophages at the site of infection or tissue injury, liver cells are stimulated to increase synthesis of CRP. Within five to six hours of the initial insult, plasma CRP concentration begins to rise rapidly to a maximal peak concentration at around 48 hours. In the case of bacterial infection, concentration can increase up to 10 000 fold¹. As the stimulus for increased production ceases, plasma CRP concentration falls rapidly to normal concentration; CRP has a half-life in plasma of 19 hours, meaning plasma ­concentration is halved every 19 hours, once the stimulus for increased production is removed.

The physiological function of CRP is as a contributor to the body’s overall innate defences against microbial attack. In this regard it is potent activator of the ­complement cascade, which leads to phagocytosis and bacterial destruction.

Laboratory measurment

Patient preparation

No particular patient preparation is necessary.

Sample timing

Blood for CRP may be taken at any time of the day, ideally at a time that allows immediate transport to the laboratory. However CRP is stable and samples can be stored at room temperature or in a sample fridge for up to 48 hours before being processed in the laboratory.

Sample requirements

Around 5 ml of blood is required for CRP measurement. Analysis can be performed on either serum or plasma. If local policy is to use serum the blood must be ­collected into a plain tube (without additives). If local policy is to use plasma blood must be collected into a tube containing the anticoagulant lithium heparin.

Interpretation of results

Approximate reference range

Causes of increased CRP

General considerations

Increase in both CRP and ESR are due to an acute phase reaction (APR) to tissue injury, infection, inflammation or malignancy. ESR is an indirect measure of APR (because it measures an effect of APR, namely the increased rate of red cell ­sedimentation) whereas CRP is a more direct measure. Still, in general terms, the conditions that give rise to a raised ESR, also give rise to an increase in CRP; like ESR, CRP is a non-specific indicator of tissue injury, infection, inflammation and malignancy.

CRP has some advantages over ESR. These include:

• Rapidity of response – CRP rises within hours of an insult (e.g. infection); ESR is slower to respond, over a period of days and weeks rather than hours.
  
• Sensitivity – CRP is generally a more sensitive test of inflammation; minimal inflammation might not be detected if only ESR is measured.
  
• Specificity – CRP is not affected, as ESR is by abnormality in red cell numbers and shape. So that, for example, CRP remains normal in patients whose sole problem is anaemia. ESR is raised in this condition, giving false evidence of inflammation or infection etc.

Infection

CRP is raised following infection. Bacterial infection is associated with highest concentration (in the range 80–1000 mg/L). The rise is more modest during viral infection (10–20 mg/L). This difference has been utilised clinically, for example, in identifying the cause of meningitis (bacterial versus viral). Measurement of CRP has proven useful for early identification of bacterial infection in a variety of ­clinical contexts. For example, detection of postoperative infection and infection among patients being cared for in intensive care (a group at particularly high risk of ­hospital acquired infection).

Inflammation

CRP is a very sensitive indicator of severity of inflammation. Highest concentration (>200 mg/L) indicates acute inflammation, for example, during the active phase of chronic conditions such as rheumatoid arthritis and Crohn’s disease. With few exceptions, all conditions with an inflammatory component are associated with increase in CRP concentration; the magnitude of the increase reflecting severity. The test is very helpful in providing objective evidence of the therapeutic effect (or the lack of it) of treatment for inflammatory disease.

Tissue injury

Tissue injury, whatever its cause, is associated with increased CRP concentration. Severity correlates with concentration so that particularly high concen­trations (>500 mg/L) might be evident following severe trauma or major surgery. The ­necrosis of heart muscle associated with myocardial infarction causes increase.

Clinically useful anomalies

As we have seen the normal response to active inflammatory disease is an increase in plasma CRP concentration. For reasons that remain unclear that response is either significantly lower in magnitude or entirely absent in a few inflammatory conditions. This has proven diagnostically useful because there are very few inflammatory conditions in which ESR is significantly raised (reflecting an inflammatory process) but plasma CRP is only slightly raised or even normal. One of these conditions is systemic lupus erythematosus (SLE or lupus), a relatively common chronic autoimmune disease that predominantly affects women of child-bearing age. Joint inflammation similar to that seen in rheumatoid arthritis is a common feature of lupus. When this inflammation occurs in the lupus patient it is accompanied as expected by a marked increase in ESR. However in contrast to most other inflammatory conditions, the plasma CRP remains resolutely normal. The combination of raised ESR and normal CRP is a useful diagnostic feature of SLE.

A similar combination of results (raised ESR and normal or only slightly raised CRP) is typical of the inflammatory bowel disease, ulcerative colitis. This feature distinguishes it from the only other inflammatory bowel disorder Crohn’s disease, in which the increase in ESR and CRP are equal. The difference can be diagnostically helpful.

CRP and cardiovascular disease

The development of highly sensitive assays for CRP measurement that are capable of detecting concentration well below 10 mg/L (the limit of original assays used for CRP testing) has revealed that the mean concentration of plasma CRP in ­apparently healthy individuals is around 0.8 mg/L with a range of 0.01–10 mg/L. Many studies conducted over the past decade have demonstrated that CRP within this normal range predicts future risk of cardiovascular disease. The higher the plasma CRP concentration the greater is the risk of cardiovascular disease². The only blood test currently used to assess an individual’s risk of cardiovascular disease is blood cholesterol measurement (see Chapter 8). It seems possible that in the future ­measurement of plasma CRP might also be used in this way. Indeed at least one authoritative guideline³ recommends this use of the high-sensitivity CRP test, and states that statin (cholesterol lowering) therapy aimed at reducing an individual’s risk of cardiovascular disease should be offered to all those with a CRP >2 mg/L, irrespective of their blood cholesterol level.

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