LABORATORY INVESTIGATIONS CONTENTS

Written by David Woods

BSC (Hons) MPharm FNZHPA MPS

GENERAL PRINCIPLES – THE REFERENCE RANGE

CREATININE AND ESTIMATION OF RENAL FUNCTION UREA ELECTROLYTES – SODIUM, POTASSIUM, CALCIUM LIVER FUNCTION TESTS FULL BOOD COUNT MISCELLANEOUS TESTS CREATINE KINASE (CK)

ERYTHROCYTE SEDIMENTATION RATE (ESR) C-REACTIVE PROTEIN

CASE STUDIES

INTRODUCTION

Interpretation of laboratory investigations is an important skill in medicines management. Knowledge and competence in this discipline allows the pharmacist the opportunity to provide effective advice in monitoring drug therapy, identification of inappropriate therapy or untreated problems, and of course identification and management of adverse drug reactions. In answering drug information question it is often necessary to obtain and interpret laboratory investigations. For example, if a drug is suspected of causing liver disease it is necessary to interpret the results of liver function tests.

1

GENERAL PRINCIPLES – THE REFERENCE RANGE

Laboratory values are reported back in relation to a “normal” or reference range. The term “reference range” is more appropriate as patients can have results outside the range and still be disease free i.e. normal (see below). For most parameters the reference range is derived from populations of disease free, asymptomatic individuals. In statistical terms the range of values is represented as a normal distribution. The reference range is plus or minus 2 standard deviations from the mean value (see Figure 1). Taking potassium as an example (reference range 3.5 – 5 mmol/L), this means that 95% of disease free individuals will have a value within this range but that 5% will have a value of less than or greater than the reference range. Therefore, an abnormal value may not necessarily indicate a pathological state. Furthermore, many disease states will also be represented by a normal distribution of laboratory values, and as the population values of normal and abnormal values may overlap there is the possibility of false positive and false negative results (see Figure 2). Sampling error can also cause erroneous results. In the case of potassium, haemolysed red cells in the sample can cause a high result by releasing intracellular potassium into the sample. The ideal laboratory test would definitively differentiate between patients with disease and those without (Figure 3). This is rarely the case due to the natural variation within “normal” and diseased patients. Sensitivity and Specificity A test that is highly sensitive will show a positive result in a high percentage of patients who actually have the disease. A test is 100% sensitive if it shows a positive result in every patient who has the disease. In the case of temporal arteritis the ESR is a very sensitive test as a negative result virtually rules out the disease. The inflammatory nature of the disease causes an increase in ESR. As the ESR can be increased in many inflammatory diseases it is not very specific for temporal arteritis and the result has to be used in conjunction with other tests and symptoms. Accuracy is the proximity of the measured value to the true value and precision refers to the reproducibility of the test assay. All these factors emphasise the skills and considerations required in the interpretation of laboratory results. It is also clear that acting on a single result may not be appropriate in many situations so it is important to repeat measurements in conjunction with other signs and symptoms. Please note that the reference ranges may vary slightly from those quoted in other sources or from your local diagnostic laboratory.

2

Figure 1. The Reference Range

‘Normal’ Subjects

TestValue

-2SD +2SD

5 mmol/L3.5mmol/L

The reference range extends plus or minus 2SD either side ofthe mean value, i.e. 95 % of disease free individuals

Figure 2. False +ve and false –ve results

‘Normal’ Subjects

TestValue

-2SD +2SD

5 mmol/L3.5mmol/L

‘Abnormal Results’

‘Normal results may be associated with disease and ‘abnormal results’ may occur in disease free individuals.

Figure 3. The Ideal Test

‘Normal’ Subjects

TestValue

-2SD +2SD

‘Abnormal Results’

3

CREATININE AND ESTIMATION OF RENAL FUNCTION AND ESTIMATION OF RENAL FUNCTION Creatinine ( 80 -150 µmol/L ) Creatinine is produced by skeletal muscle and is filtered by the kidney tubules and then excreted in the urine. As creatinine is not actively secreted or reabsorbed in the kidneys (i.e. filtration is passive) measurement of serum creatinine can be used to estimate Glomerular Filtration Rate (GFR) which is a measure of renal function. To estimate GFR the creatinine clearance (CrCl) is calculated by using the Cockcroft and Gault equation (see below). This equation takes into consideration serum creatinine and factors that influence renal function (age) and creatinine production (muscle mass). CrCl is usually expressed in mL/minute or mL/hour. Normal renal function in a young healthy adult is 100 – 120 mL/min but this declines with age. For example, 50% of people aged 65 will have a CrCl of less than 80 ml/min and 50% of people aged 70 will have a CrCl of less than 60 mL/min. The degree of renal impairment is classified according to the Creatinine Clearance (Table 1).

Table 1. Classification of renal impairment

Grade GFR Creatinine Clearance mL/min

*Serum Creatinine µmol/L

Mild 20 - 50 150 - 300 Moderate 10 - 20 300 – 700 Severe > 10 > 700 *These values of serum creatinine are a very crude estimate of the respective CrCl as creatinine is influenced by muscle mass and physical activity. It is always preferable to make a better estimate of renal function by calculating CrCl from the Cockcroft and Gault equation. Be wary of drug data sheets that suggest dosage adjustments based solely on measured serum creatinine. Relevance of creatinine clearance calculation in medicines management. Many drugs or their active metabolites are excreted via the kidneys so when CrCl declines doses may have to be reduced to prevent toxicity.

• Use estimated CrCl to guide dosage of drugs that require adjustment in renal impairment.

• Use CrCl rather than serum creatinine. • For obese patients use lean or ideal body weight rather than actual

body weight in the Cockcroft and Gault equation • It is especially important to estimate renal function in the elderly. You

can assume that all patients over 60 years of age have some degree of renal impairment. Patients with comorbidities, e.g. diabetes, CHF or pre-existing renal disease are especially at risk.

4

• Important drugs that require dosage adjustment include; lithium, fibrates, digoxin, ACE inhibitors, NSAIDs, long acting sulphonlyureas, allopurinol, metformin and colchicine. There are many others.

• Serum creatinine may not accurately estimate CrCl in rapidly changing renal function as serum Cr takes a few days to reach a new steady state. In these situations several serial measurements of serum Cr should be made.

• For information on dose adjustment for specific drugs see the drug’s data sheet, BNF section on dosing in renal impairment or the NZ Dispensing Guide. There is also an excellent on-line resource (see Appendix 1).

COCKROFT and GAULT EQUATION

CrCl = (140 - age) x Wt (kg) x F

Cr

Units are mL/minute Cr = serum creatinine in µmol/L F = 1.23 for males, 1.04 for females

(takes into account the greater muscle mass in males) Please note that there are different versions of this equation depending on the units that are used to report serum creatinine. The principles are the same. This equation takes in to consideration age (declining renal function), body weight (relates to muscle mass and therefore creatinine production), and of course serum creatinine. The equation is only an estimate of renal function and significant under-estimation of CrCl is possible. For example, consider an elderly, bedridden patient with significant muscle wastage. Creatinine production will be reduced due to reduced muscle mass and inactivity. A lower than expected serum creatinine will tend to over estimate renal function in the equation above. If actual body weight is used for significantly obese patients the amount of creatinine-producing muscle mass is over estimated and again renal function may appear better than it really is. In the latter case a correction is made by using the patient’s lean or ideal body weight which equates better to skeletal muscle mass.

5

UREA

Urea ( 3 – 8 mmol/L) The breakdown of exogenous (i.e. food) and endogenous (e.g. muscle) amino acids in the body generates large amounts of ammonia, which is toxic and is rapidly converted to urea in the liver. Urea is excreted in the urine but is not a sensitive marker of renal function as the serum urea or BUN (blood urea nitrogen) is influenced by other factors. BUN is increased by a high protein intake, upper GI bleeding (protein from digested blood increases urea formation) and dehydration. BUN can be decreased by severe starvation and severe liver failure due to decreased production of urea. BUN concentrations of greater than 10 mmol/L usually indicate severe renal disease or reduced renal blood flow due to shock, fluid loss or dehydration. In severe renal failure BUN concentrations are markedly increased (uraemia) causing symptoms such as fatigue, malaise and pruritus (itching).

ELECTROLYTES Before discussing some individual electrolytes it is necessary to briefly review some important principles and terminology. Fluid volume in the body is comprised of three compartments. A delicate equilibrium exists (fluid homeostasis) between the compartments to maintain electrolyte concentrations, volume and osmotic pressure (Table 2). Intracellular volume : Fluid volume within cells. Extracellular volume : Fluid outside the cells; includes interstitial fluid and intravascular fluid. Interstitial volume : Fluid between tissue or cells; extracellular fluid excluding the fluid in blood vessels.

Table 2. Body Fluid Compartments

Intracellular Fluid Extracellular Fluid 40–45% of body wt Volume about 30 L Main electrolytes: potassium and phosphate

Interstitial Fluid 11 – 15 % of body wt. Volume about 10 L sodium and chloride

Intravascular Fluid 5% of body wt. Volume about 3 L sodium and chloride

↔ ↔

6

Numerous compensatory mechanisms are involved in fluid homeostasis. Three important hormones are aldosterone, ADH and renin. All these are inter-related in the renin-angiotensin-aldosterone system or RAAS (Figure 4). Aldosterone is excreted by the adrenal cortex in response to low circulating blood volume, low serum sodium, high serum potassium and Angiotensin II. It increases sodium reabsorption from the distal tubules in the kidney in exchange for potassium, which is then excreted in the urine. Antidiuretic Hormone (ADH or vasopressin). The hypothalamus contains cells that ‘read’ the osmolality of blood. If the osmolality is high (high serum sodium or low serum water) the hypothalamus signals the posterior pituitary to release ADH, which increases reabsorption of water from collecting tubules in the kidney. Renin Renin is released from the kidney in response to reduced renal blood flow or pressure or low serum sodium

Figure 4. The Renin-Angiotensin-Aldosterone (RAAS) system

kidney Na

+

renin

angiotensinogen angiotensin I angiotensin II

Angiotensin Converting Enzyme ACE Inhibitors block here

vasoconstriction

blood pressure

adrenal cortex

brain thirst

ADH

aldosterone

Na +

reabsorption plasma K

+

renal blood flow or pressure

For example, if intravascular blood volume is reduced by acute blood loss, the secretion of aldosterone is increased, which then increases the reabsorption of sodium in the kidney tubules. Along with the sodium there is also an increase in water reabsorption which helps to correct fluid volume and maintain renal function. The compensatory mechanisms are complex and inter-related and in some disease states they can aggravate the situation rather than resolve it. Take heart failure for example. Perfusion of blood through the kidneys is reduced due to reduced cardiac output and the kidneys respond by increasing renin secretion. Renin (via angiotensin I and angiotensin II) stimulates aldosterone release from the adrenals. This causes retention of sodium and water, which aggravates the pre-load on the heart.

7

Sodium Reference range 136 – 145 mmol/L Sodium is the major extracellular cation and has an important role in controlling the osmotic pressure of extracellular fluid. Water and sodium homeostasis are closely linked and any measurement of serum sodium concentration needs to be considered in relation to water homeostasis. Hyponatraemia or low serum sodium is frequently encountered in clinical practice and there are many causes. The descriptions below stress the importance of additional investigations when hyponatraemia is detected.

Different presentations of hyponatraemia

Low total body sodium. Both sodium and water are lost but more sodium is lost relative to the water loss. e.g. diuretics, prolonged vomiting and diarrhoea. These patients are usually hypovolaemic and show signs of dehydration. Net sodium loss also occurs in salt-losing conditions such as Addison’s Disease and is often associated with hypotension. Normal total body sodium The usual cause is water excess e.g. in Syndrome of Inappropriate Anti-Diuretic Hormone secretion (SIADH). In this condition, which can be caused by drugs such as SSRIs, there is either secretion of ADH without the usual “switch on” mechanism or increased kidney sensitivity to ADH. The result is increased water retention, which then dilutes the sodium in the blood. Body stores of sodium are not reduced but there is an apparent hyponatraemia. These patients don't usually have signs of volume change. In patients with SIADH the sodium concentration of the urine will be increased (i.e. urine is concentrated) due to reduced water excretion. Measurement of urine sodium concentration and osmolality are important additional diagnostic tests in patients with hyponatraemia. Raised total body sodium This is a relative dilutional hyponatraemia. In conditions such as CHF, both water and sodium are retained due to compensatory mechanisms. More water is retained relative to sodium so there is still a dilutional effect. These patients will look oedematous. The key to diagnosis is assessment of the patient's volume status, symptoms and how long the sodium has been low. Signs and symptoms of hyponatraemia include hypothermia, seizures, decreased tendon reflexes, muscle cramps, lethargy, nausea, disorientation and agitation. Hypernatraemia (serum sodium > 145 mmol/L) is much less common than hyponatraemia. The most common causes are an impaired thirst mechanism, which can occur following a stroke, or excessive water loss relative to sodium (via skin, respiration or renal). Hypernatraemia can also occur in the presence of low, normal or high total body sodium.

8

Potassium

Reference range 3.5 – 5 mmol/L Potassium is the main cation of the intracellular space and is pivotal in controlling the osmotic pressure of the intracellular fluid. The serum concentration is low compared with the intracellular concentration of around 140 mmol/L. Obviously haemolysed red cells in a blood sample can give an erroneously high serum potassium concentration. The major role of potassium is in regulating nerve and muscle excitability. Hypokalaemia Is due to true deficit or apparent deficit. In true deficit there is actual loss or reduced intake of potassium, for example, as in poor diet, anorexia nervosa, gastrointestinal loss or drugs such as diuretics, corticosteroids and liquorice abuse. Apparent deficit is when body content is normal but there is a shift of potassium into the cells. Insulin, alkalosis and β2-adrenergic stimulation can cause this intracellular shift. Thus excessive insulin administration and high doses of salbutamol can cause hypokalaemia due to intracellular shifting and an apparent deficit. Hypokalaemia results in cardiac arrhythmias, cramps and muscle weakness and increases the risk of digoxin toxicity. Hyperkalaemia As with hypokalaemia, hyperkalaemia is the result of an apparent or true imbalance of potassium. True potassium excess is due to increased intake or generation (e.g. haemolysis, muscle crush injuries, burns, potassium supplements) or decreased output (e.g. renal failure, potassium sparing diuretics, ACE inhibitors and Addison’s Disease). Apparent excess is due to extracellular shifting of potassium and can be caused by metabolic acidosis. Symptoms of hyperkalaemia include cardiac disturbances including cardiac arrest in severe cases, and muscle weakness. Patients taking ACE inhibitors, spironolactone and amiloride can be at risk of hyperkalaemia especially if renal function is impaired.

9

Calcium

Most of the calcium in the body is found in bone and only a very small proportion (about 0.0005%) is found in the serum where it is in three forms; bound to plasma proteins, mainly albumin (40%), complexed with citrate and phosphate (6%) and as free calcium ions (54%). The free ions are physiologically active. Binding of calcium to albumin is influenced by blood pH. Alkalosis increases binding and reduces free calcium. Conversely, acidosis reduces binding and increases the availability of free calcium ions. The other important factor which influences free calcium concentration is the albumin concentration. If serum albumin is low availability of free calcium is increased. As serum calcium is reported as total calcium (i.e. bound and unbound) the free calcium concentration must be corrected for albumin concentration and interpreted in the light of any changes in blood pH. Examples: 1. A patient with low albumin due to liver disease may have a normal total calcium concentration but when this is corrected for low albumin a high result is obtained and the patient may be at risk of hypercalcaemia. 2. A patient with low blood pH (acidosis) may have symptoms of hypercalcaemia with a normal serum calcium concentration. This is due to reduced binding of calcium to albumin.

10

LIVER FUNCTION TESTS (LFTS)

Transaminases Alanine transaminase (ALT) < 35 U/L Aspartate transaminase (AST) < 40 U/L AST (previously SGOT) and ALT (previously SGPT) are the transaminases. You only need to check a few drug data sheets to see phrases such as “increases in transaminases have been reported” or “increases in transaminases are usually transient and asymptomatic”. Both are found in large concentrations in the liver cells (hepatocytes) and muscle cells, and in lesser amounts in the heart, pancreas, and kidney. They are released from the liver into the blood when hepatocytes are damaged. Serum AST is increased in many conditions including liver disease, muscle trauma, myocardial infarction and surgery. ALT is more specific to the liver and is increased in liver disease but to a lesser extent than AST in other conditions. In myocardial infarction serum AST is increased due to damaged cardiac muscle cells whereas any increase in serum ALT is minimal. Transaminases may be raised in all types of acute and chronic liver disease including viral and non-viral hepatitis. They are usually markedly increased in acute drug toxicity (e.g. paracetamol poisoning) and acute hepatitis. Increases in transaminases are often less dramatic in chronic liver disease or longstanding damage. The AST/ALT ratio is typically greater than 1 in alcoholic liver disease and less than 1 in non-alcoholic liver disease. Unfortunately serum concentrations of AST and ALT cannot be used to measure liver function in order to estimate drug dosage reductions. There is no real correlation between the magnitude of transaminase elevation and prognosis. For example, methotrexate can cause complete liver failure with minimal elevation of AST and ALT. Patients with acute increases of > 1000 U/L due to drug toxicity may recover completely. As with all laboratory investigations the pattern of sequential results and the other associated symptoms or values are very important in identifying the cause.

Alkaline phosphatase (ALP) Reference range: varies with age but for adults aged 20 – 50 it is about 25 – 110 U/L. Alkaline phosphate is found mainly in the bone and liver but is also found in smaller quantities in the intestine, placenta and the renal tubules.

11

Levels of the enzyme are raised in many types of liver disease. Normally ALP is secreted into bile and excreted with the bile into the intestine. Biliary obstruction stimulates the production of ALP in the hepatocytes. Since the elimination pathway is blocked, significantly raised plasma levels of enzyme appear where there is intrahepatic or extrahepatic cholestasis. Alkaline phosphatase is not specific to the liver and is raised in many other conditions including bone disease, growth spurts in adolescents, bone healing and pregnancy. Some of these are obviously not pathological but raised ALP in combination with other factors e.g. raised AST and ALT may indicate disease. If the source of the raised ALP is not due to liver disease or physiological process, a bone disease such as osteomalacia, Paget’s Disease or tumour may be suspected. Drugs such as phenytoin, phenobarbitone and lithium can cause increases in ALP.

Gamma-glutamyl transpeptidase (GGT) Reference range < 50 U/L GGT is found in large amounts in the liver, kidneys, pancreas, and prostate. Its activity rises in most types of liver disease but particularly in biliary obstruction. GGT concentration is raised to a greater extent in biliary tract disease and cholestasis compared with hepatocellular disease. GGT synthesis is increased by enzyme inducing drugs i.e. anticonvulsants and alcohol, but elevated levels will reverse on withdrawal of the drug or abstinence from alcohol.

Bilirubin Bilirubin (total) < 20 µmol/L Bilirubin (conjugated) < 4µmol/L Total bilirubin = Conjugated (direct) bilirubin + Unconjugated bilirubin (indirect) As red blood cells reach the end of their life they are taken up by the spleen and destroyed. The released haemoglobin is eventually broken down to bilirubin, which is then transported to the liver bound to albumin in its lipid soluble or unconjugated form. In the liver it is combined (conjugated) with glucuronide to make it water-soluble. Conjugated bilirubin is then excreted in the bile (via the bile duct) into the intestine where bacteria act on it to form urobilinogen. Most of the urobilinogen is excreted in the faeces but some is reabsorbed and excreted again by the liver into the gut or in the urine via the kidneys. Bilirubin concentrations are usually reported as total and then as its components; conjugated and unconjugated. There are many reasons for raised serum bilirubin (hyperbilirubinaemia) and when the total concentration reaches 35 – 70µmol/L yellowing of skin colour and sclerae (jaundice) become apparent.

12

Some causes of hyperbilirubinaemia and jaundice are: Cholestasis Bile flow is obstructed and high concentrations of conjugated bilirubin appear in the blood. The obstruction can be extrahepatic caused by inflammation, gallstones, or invading malignant tissue. Intrahepatic obstruction arises due to a problem with the liver cells (e.g. inflammation) or the bile duct within the liver. Intrahepatic cholestasis has many causes including alcoholic hepatitis, viral hepatitis, AIDS, and numerous drugs. Flucloxacillin and chlorpromazine are well known examples of drugs that can cause cholestatic hepatitis. Cholestasis often presents as a typical pattern of jaundice, raised GGT and alkaline phosphatase, but with normal or only minimally raised transaminases. However, this typical pattern is often complicated when associated with concurrent hepatocellular damage. In pure cholestasis the synthetic capacity of the liver is not reduced and conjugation of bilirubin is essentially normal but the excretion via the bile is compromised. Hepatocellular Damage Damage to the liver cells results in an inability to conjugate bilirubin. Concentrations of conjugated and unconjugated bilirubin both rise. In contrast to pure cholestatic hepatitis there is a significant increase in the transaminases (ALT and AST) when hepatocellular damage is present. However a mixed pattern can also exist with combined cholestasis and hepatocellular damage. Haemolysis Erythrocytes are destroyed at a greater rate than normal and unconjugated bilirubin accumulates in the blood and anaemia develops. The liver’s capacity to conjugate bilirubin is exceeded. In haemolysis, serum AST is increased as erythrocytes contain high concentrations of this enzyme. Haemolytic anaemia is caused by a number of congenital disorders and several drugs including methyldopa and levodopa. Red blood cells are usually protected against oxidative stress by glucose 6-phosphate dehydrogenase (G6PD). When there is a deficiency of G6PD some drugs such as nitrofurantoin and sulphasalazine can cause haemolysis. Displacement Drugs, such as sulphonamides, can displace bilirubin from the albumin binding sites. This is particularly important in neonatal jaundice or kernicterus when the displaced unconjugated bilirubin is then free to enter the CNS and cause toxicity.

Albumin Reference range 38 – 50 g/L Albumin is synthesized in the liver and serum albumin concentrations reflect the synthetic capacity and integrity of the liver. Albumin has a half-life of about

13

20 days and changes in serum concentration occur relatively slowly. A reduced serum albumin can indicate chronic liver disease. As the functional capacity of the liver is very large, albumin concentrations do not decline until significant damage has been done. Symptoms are not usually apparent until albumin concentrations are quite low. Concentrations of 20-25 g/L or lower are observed in patients with severe hepatitis or cirrhosis which leads to symptoms such as oedema in the abdomen (ascites), peripheral and pulmonary oedema. Albumin retains intravascular fluid due to its osmotic properties (oncotic pressure). When the serum albumin concentration is reduced, hydrostatic pressure (i.e. blood pressure) overcomes the oncotic pressure and forces fluid out into the interstitial spaces and body cavities. Other causes of low serum albumin (hypoalbuminaemia) include severe burns, severe psoriasis and other skin diseases, loss via the kidney (nephrotic syndrome) and through the gut in protein losing enteropathy.

Coagulation Factors (INR) The liver also synthesises coagulation factors thus there will be a prolonged prothrombin time (INR) with severe liver cell damage. Also an elevated INR may occur in severe cholestasis due to reduced absorption of Vitamin K. Prothrombin has a half-life of 2-3 days. Changes in INR occur more rapidly than albumin and therefore it is more useful in predicting the extent of hepatocellular damage in acute situations. Serum albumin, bilirubin and INR reflect the functional capacity of the liver and in combination are used to assess the severity of liver damage in the Child-Pugh score. (See website for details http://www.fpnotebook.com/GI41.htm

Relevance of LFTs in medicines management Drug induced liver disease is very common and early identification can improve the prognosis and potentially reduce morbidity and mortality. When abnormal LFTs are reported it is important to differentiate between drug-induced disease and non-drug related causes. It should also be noted that some herbal and alternative medicine products can cause liver disease. It is important to advise and ensure that appropriate monitoring of LFTs is performed with high-risk drugs, e.g. amiodarone, methotrexate, statins and sodium valproate. Guidelines and recommendations can usually be found in the drug’s data sheet. Drug datasheets can be found at http://www.rxlist.com Recognition of associated signs and symptoms and provision of patient advice are also important. For example a patient taking anti-TB drugs who complains of nausea, vomiting or abdominal pain should be followed up as they may have drug-induced hepatitis.

14

COMPLETE BLOOD COUNT (CBC)

The CBC is the most frequently ordered laboratory test. It gives information on the counts of red (RBC) and white blood cells (WBC), platelets, haemoglobin concentration, haematocrit, red cell distribution width (RDW) and RBC indices. When a differential CBC is ordered the counts of the various white cells are reported. A detailed discussion of haematopoiesis (formation of the blood cells) is outside the scope of this booklet but is summarized in figure 5. More information is available from the web sites listed in Appendix 1. A brief summary of the complex process is as follows. In the bone marrow, stem cells differentiate into lymphoid stem cells or haematopoietic stem cells. The former undergo further development in the bone marrow and eventually appear in the peripheral blood as B or T lymphocytes. The haematopoietic stem cells follow one of three development lines in the bone marrow. The proerythroblasts develop into reticulocytes (immature erythrocytes) and ultimately erythrocytes in the peripheral blood. Myeloblasts are the precursors of monocytes and promyelocytes eventually differentiate to neutrophils, basophils and eosinophils in the peripheral blood. Red Blood Cell Count (4.6 – 6.2 x 1012 cells/L for males; 4.2 – 5.4 x 1012 cells/L for females) The number of erythrocytes in a given volume of blood. The RBC count is reduced in all types of anaemia and there is also a proportional decrease in haemoglobin concentration and haematocrit. Haemoglobin (Hgb) (14 – 18g/dL for males; 12 – 16g/dL for females) The concentration of Hgb in a stated volume of whole blood. There is a direct correlation between Hgb concentration and the oxygen carrying capacity of the blood. In severe anaemia, tiredness, fatigue and even angina are consequences of reduced Hgb concentration and reduced oxygen transport capacity. Haematocrit (Hct) (0.42 – 0.52 for males ; 0.37 – 0.47 for females) Also known as the packed cell volume. The Hct is the percentage volume of blood that is made up of erythrocytes. The haematocrit is usually about three times the value of the haemoglobin concentration unless there are abnormalities in the size and shape of the erythrocytes. Hct is reduced in patients with anaemia. Red Blood Cell Indices: These specifically report the size and Hgb content of RBCs and are calculated values – not measured

15

Mean Cell Volume (MCV) 80 – 96 fL for males; 82 – 98 fL for females MCV is an estimate of the average volume of the red blood cells i.e. it represents the peak of the normal distribution curve. Therefore it is insensitive to small numbers of large or small cells. It is calculated by dividing the Hct by the RBC count. Abnormally large cells (increased MCV) are termed macrocytic which is a major feature of vitamin B12 or folate deficiencies, hence the term macrocytic anaemia. Smaller than normal cells (decreased MCV) are termed microcytic and most commonly occur with iron deficiency anaemia, which is therefore termed a microcytic anaemia. Mean Cell Haemoglobin (MCH) 27 – 33 pg/cell MCH is a measure of the average amount of Hgb per cell. It is calculated from the Hgb divided by RBC. In iron deficiency anaemia the lack of sufficient iron for Hgb production produces cells with low MCH and the RBCs appear pale or hypochromic. Mean Cell Haemoglobin Concentration (MCHC) (310 – 350 g/L) This is the Hgb divided by the Hct and gives the mean Hgb concentration of the RBCs. The MCHC is reduced in iron deficiency anaemia. Reticulocyte Count (RC) (0.5 – 2.5% of RBCs) Reticulocytes are immature RBCs that are released into the circulation and rapidly differentiate into RBCs over the first 24 - 48hours of their lifespan. The reticulocyte count is approximately 1% of the circulating RBC population and this reflects the daily RBC replacement rate. RC is increased in response to blood loss and haemolysis and is decreased in untreated iron deficiency and B12 or folate deficiency anaemia. When iron deficiency anaemia is treated with iron salts the RC increases in response to stimulation of haematopoiesis. Other important haematological tests relevant to anaemias Serum Vitamin B12 (30 - 830 pmol/L) Vitamin B12 deficiency is caused by inadequate dietary intake and defective production of intrinsic factor, which is required for absorption in the stomach. Deficiency caused by inadequate diet is rare and occurs only in those who do not eat any animal products including milk and eggs. Deficiency of intrinsic factor (pernicious anaemia) is the most common cause and can be due to an autoimmune disease or gastric surgery. Symptoms of B12 deficiency include loss of appetite, sore tongue (glossitis), constipation or diarrhoea and CNS effects such as tingling and numbness in extremities, loss of limb co-ordination and tremors.

16

Serum Folate (8.5 – 30 nmol/L) Within 5 weeks of inadequate folate intake, the serum levels fall to below normal but folate concentrations in RBC take longer to fall. As serum folate levels can fluctuate from day to day depending on dietary intake, erythrocyte folate levels are a better indicator of longstanding deficiency. Folate deficiency can be caused by inadequate dietary intake, malabsorption or impaired conversion to the active form in-vivo. Anti-folate drugs such as methotrexate can cause the latter. Signs and symptoms of folate deficiency are similar to those of B12 deficiency but diarrhoea is more common than constipation. Weakness, breathlessness, headache and palpitations also occur. Both vitamin B12 deficiency and folate deficiency cause macrocytic (megaloblastic anaemia) and, to some extent, similar symptoms. Administration of folic acid will alleviate some of the common symptoms but will not resolve the serious, progressive neurological problems caused by B12 deficiency. Consequently the underlying deficiency must be identified before deciding on appropriate treatment. Serum Iron (50 – 100 µg/dL) This measures iron that is bound to the transporter protein transferrin. Serum iron concentration is reduced in iron deficiency anaemia. The value represents about one third of the TIBC of transferrin (see below). Total Iron Binding Capacity or TIBC (250 – 410 µg/dL) The TIBC measures the iron binding capacity of transferrin. In iron deficiency anaemia the synthesis of transferrin is increased. This leads to an increase in the TIBC as the percentage saturation is decreased. In a normal subject with a serum iron of 80 µg/dL and a TIBC of 240 µg/dL the percentage saturation is 33%. If serum iron is reduced to 25 µg/dL with a corresponding increase in TIBC to 500 µg/dL the percentage saturation is only 5%. When transferrin saturation is reduced to 15% iron deficiency anaemia is usually present. Ferritin (10 – 20 µg/L) This is the iron storage protein and it is markedly reduced in iron deficiency anaemia. However, in anaemia of chronic disease the ferritin concentration is normal or slightly increased. There are many types of anaemia and diagnosis can be complex due to mixed presentations. Table 3 summarises some important anaemias and their features. Apart from iron deficiency anaemia it is important to have a working knowledge of macrocytic anaemia (due to folate or B12 deficiency – described above) and anaemia of chronic disease. The latter occurs in association with some chronic diseases and infections (Table 3) and is usually normocytic and normochromic although it can be microcytic. The pathophysiology is thought to involve some malfunction of iron utilisation and mobilisation. Compared with iron deficiency anaemia there is no decrease in ferritin concentration.

17

Table 3. Summary of Laboratory Assessment of Anaemia

Anaemia Iron Deficiency Chronic Disease Macrocytic (megaloblastic)

Causes Blood loss, poor dietary intake

Chronic disease such as RA, inflammatory conditions, malignancies, chronic infections e.g. TB

Vitamin B12 deficiency Folic acid deficiency Drugs (folic acid deficiency)

RBC, Hgb, Hct Decreased

Decreased Decreased

MCV Decreased

Decreased or no change Increased

MCH Decreased Decreased or no change

Increased

MCHC Decreased

Decreased or no change

No change

Reticulocytes Decreased or no change

Decreased or no change Decreased or no change

Serum Iron Decreased

Decreased No significant change

TIBC Increased

Decreased No significant change

Ferritin

Decreased No change or increased No significant change

Figure 5. Haematopoiesis

(red boxes indicate usual presence in peripheral blood)

B Lymphocyte T Lymphocyte

Plasmacyte

Lymphoblast

Lymphoid Stem Cell

Erythrocyte

Reticulolcyte

Erythroblast

Proerythroblast

Monocyte

Promonocyte

Myeloblast

Neutrophil

Band Neutrophil

Metamyelocyte

Myelocyte Basophil Eosinophil

Promyelocyte

Haematopoietic Stem Cell

Pluripotent Stem Cell

18

White Blood Cell Count White blood cells (leukocytes) play a crucial role in fighting infection and immunological response. Many drugs can adversely effect the production and function of white cells and such adverse drug reactions (blood dyscrasias) are a major cause of drug related morbidity and mortality.

The total WBC count gives the total number of all white cell types. As such this is non-specific as it could indicate a predominant rise or fall in one particular cell line. A differential WBC count gives more information. The differential count is often expressed as a percentage of the total count. Table 4 gives the usual (reference range) differential percentages of the various white cell types. An absolute count of individual cell types will define specific abnormalities. For example a total WBC is reported as 3.6 x 109 cells/L (low) and the differential indicates 25% neutrophils, which is also low. An absolute count of the neutrophils reports 0.9 x 109 cells/L which is defined as a neutropenia ( i.e. < 1.5 x 109 cells/L) see Table 4.

Numerous drugs can cause neutropenia (e.g. clozapine, carbamazepine, methotrexate), which puts the patient at severe risk of infection and can be fatal. Agranulocytosis is when the neutrophils and similar white cell lines are completely ablated. Early detection of neutropenia by regular white cell counts is recommended for many drugs (e.g. clozapine) but with some drugs the WBC count falls so quickly that regular monitoring may not detect it. A patient who is taking any drug that can cause neutropenia or agranulocytosis should be advised to report signs of infection, such as sore throat, which may indicate a blood dyscrasia.

Pancytopenia is when the count of all circulating cells is reduced, including red cells, and indicates bone marrow toxicity or aplastic anaemia.

Platelets 150 – 400 x 109 cells/L

Most platelets are formed in the bone marrow from megakaryocytes but the lungs and other tissues can also form platelets. Platelets are involved in haemostasis and the coagulation cascade.

Thrombocytopenia is a reduction in platelets to less than 150 x 109 cells/L. As the platelet count decreases spontaneous bleeding becomes more likely but the risk is not directly proportional to the degree of thrombocytopenia. A count of 0 – 20 x 109 cells/L is severe thrombocytopenia which is life threatening. Acute infections and numerous drugs including heparin, phenytoin, sulphonamides, phenobarbitone and carbamazepine can cause thrombocytopenia. Note that anticoagulants such as warfarin do not work by reducing platelet counts but on the coagulation cascade. This means that a patient with an increased INR will usually have a normal platelet count and a patient with thrombocytopenia will have a normal INR. Thrombocytosis refers to an increase in the platelet count above 450 x 109 cells/L. Transient thrombocytosis is commonly caused by blood loss, acute infection (including viral), surgery, trauma and inflammatory disorders.

19

Table 4 White Blood Cell Count and common causes of abnormal results.

Reference Range (as % of total WBC)

Abnormal Results (with absolute count and examples

of causes White Blood Cell Count WBC

4.8 – 10.2 x 109 cells/L 100 %

If WBC is increased or decreased a differential count is required to identify which cell line(s) are involved.

Neutrophils

45 – 75 %

Neutrophilia > 12 x 109 cells/L e.g. acute bacterial infection Neutropenia < 1.5 x 109 cells/L e.g. medications, overwhelming acute bacterial infection

Eosinophils

0 – 4 %

Eosinophilia >0.35 x 109 cells/L Allergies, asthma, allergic drug reactions

Basophils

0 – 1 %

Basophilia > 0.3 x 109 cells/L Chronic inflammation, leukaemia

Monocytes

2 – 8 %

Monocytosis > 0.8 x 109 cells/L Recovery stage of acute bacterial infection, TB, endocarditis, leukaemia

Lymphocytes

20 – 40 %

Lymphocytosis > 4 x 109 cells/L Viral infections, lymphoma, TB Lymphopenia < 1 x 109 cells/L HIV, corticosteroids, radiation exposure

20

MISCELLANEOUS TESTS

Creatine kinase (CK) CK is found in skeletal muscle, heart muscle, smooth muscle, brain and other tissues. CK concentrations increase when these tissues are traumatised as in myocardial infarction, shock, muscle injury and surgery. Even physical activity (e.g. a long run) or intramuscular injection can produce mild increases. There are three sub-types of CK (BB, MM and MB). The predominant sub-type in the serum is MM as this comes mainly from skeletal muscle. Heart muscle contains more of the MB sub-type and concentrations are increased following myocardial infarction. A number of drugs can cause an increase in CK. This can be either mild and symptomatic or associated with drug-induced damage to the muscles. For example, the statins (e.g. simvastatin, atorvastatin, pravastatin) cause mild increases in CK in many patients but occasionally the increases are marked and associated with severe muscle breakdown (rhabdomyolysis) which can lead to renal failure. Patients on statins who complain of muscle aches and pains should have their CK levels checked. For guidance on monitoring CK for individual drugs, especially stains, refer to the drug’s data sheet.

Erythrocyte Sedimentation Rate (ESR) Normal Range 1 – 15 mm/hr for males

1 – 20 mm/hr for females ESR increases with age. Erythrocytes usually settle slowly in plasma but when aggregation occurs the sedimentation rate is increased. Aggregation is usually prevented by a net negative charge on the red cell membrane, i.e. the cells repel each other. Some proteins, including acute phase reactants (released during inflammatory processes) are positively charged and can neutralise the negative charge on the erythrocyte. Consequently aggregation occurs and the ESR increases. ESR is increased in diseases with an inflammatory component including rheumatoid arthritis, osteomyelitis and temporal arteritis. This test is not used to diagnose disease in isolation but is useful in monitoring inflammatory disease processes and response to drug therapy. The ESR is often significantly raised during an active exacerbation of disease but falls when inflammation or disease severity lessen.

C- Reactive Protein (CRP) C- reactive protein is an acute phase response plasma protein. CRP concentration increases in response to tissue injury and infection and assists phagocytes to bind to and activate the complement system in the immune response. CRP concentration is a measure of the activity of the immune system. CRP is not disease specific and is used and interpreted in a similar way to ESR.

21

APPENDIX 1

Useful Web Sites Medlab Handbook http://www.dml.co.nz/clin_handbook.html Very comprehensive resource that can be viewed online or downloaded for use off-line. Includes notes on interpretation. Global RPh. Interpretation of Lab tests. http://www.globalrph.com/labinter.htm Short notes on interpretation of common laboratory investigations. Also links to relevant sections of the Merck Manual. Interpretation of Lab Test profiles by Ed Uthman M.D. http://web2.iadfw.net/uthman/lab_test.html Notes on interpretation of laboratory tests including full blood count. Manual of Use and Interpretation of Pathology Tests: Third Edition. Royal College of Pathologists of Australia. http://www.rcpa.edu.au/pathman/test_lis.htm Select your test from the alphabetical menu Creatinine Clearance calculator http://www.globalrph.com/crcl.htm Type in the patient’s details (ensure correct units) and calculate the estimated creatinine clearance. Drug Prescribing in Renal Failure: Dosing Guidelines for Adults. Fourth Edition. Aronoff GR, Berns JS, Brier ME, Golper TA, Morrison G, Singer I, Swan SK, Bennett WM. American College of Physicians. On-line version; http://www.kdp-baptist.louisville.edu/renalfailure/ Blood Cells and the CBC http://web2.iadfw.net/uthman/blood_cells.html

22

APPENDIX 2

Demonstrative Cases These short cases will help to understand the principles that you have learned. The discussion of each case is given below. Case 1 Mr. R has an AST concentration of 420 U/L and an ALT concentration of 230 U/L. GGT concentration is 467 U/L MCV is increased (i.e. enlarged white cells) but folic acid and B12 concentrations are normal. Case 2 Mrs D is a 75 year is old woman with rheumatoid arthritis and nerve pain. Medications; diclofenac 25 – 50 mg PRN for pain and carbamazepine 200 mg TID for nerve pain. Basic lab tests report the following.

GGT 231 U/L Hb 9 g/dL

The blood film shows a large number of macrocytic cells. She has been put on oral iron tablets and folic acid 5 mg daily. Case 3 A 75 year old woman who weighs 60 kg has a Serum Cr of 180 µmol/L What dose of allopurinol should be prescribed? Case 4 An man aged 70 complains of weakness and dizziness. He has been taking paroxetine 20 mg daily for about 4 weeks. There are no other recent medication changes. A screen of urea and electrolytes is performed. The only significant result is a serum sodium concentration of 121 mmol/L. =========================================================== Discussion of Case 1 Mr. R has an AST concentration of 420 U/L and an ALT concentration of 230 U/L. GGT concentration is 467 U/L. MCV is increased (i.e. enlarged white cells) but folic acid and B12 concentrations are normal. An AST:ALT ratio of >1 in combination with an increased GGT often indicates alcoholic liver disease. This theory is strengthened by the observation of macrocytosis (enlarged white cells), which is not due to folate or B12 deficiency. Investigation of Mr. R’s alcohol drinking habits would also be helpful.

23

Discussion of Case 2 Mrs D is a 75 year is old woman with rheumatoid arthritis and nerve pain. Medications; diclofenac 25 – 50 mg PRN for pain and carbamazepine 200 mg TID for nerve pain. Basic lab tests report the following.

GGT 231 U/L Hb 9 g/dL

The blood film shows a large number of macrocytic cells. She has been put on oral iron tablets and folic acid 5 mg daily. GGT in isolation may indicate enzyme induction due to the carbamazepine but other LFTs should be measured. Also question alcohol intake. Mrs D is anaemic but what is the cause ? The diclofenac could be causing blood loss so it would be useful to check for blood in the faeces. However if iron deficiency anaemia (IDA) is significant undersized red blood cells (microcytic) with a low haemoglobin concentration (hypochromic) would be expected. Mrs D has a chronic disease (RA) but anaemia of chronic disease is normocytic or microcytic. If serum ferritin were measured a low result would tend to support the diagnosis of IDA. There is also a suggestion of megaloblastic anaemia due to the observation of large (macrocytic cells). It would also be important to check serum folate and B12. If folate is given in unrecognised B12 deficiency progressive, severe neurological symptoms may be masked. This case could be very complex and the cause of anaemia could be a combination of types. However, it emphasises the importance of interpretation and further investigations. Discussion of Case 3 A 75 year old woman who weighs 60 kg has a Serum Cr of 180 µmol/L What dose of allopurinol should be prescribed? Using the Cockcroft and Gault equation a CrCl of about 23 ml/min is a calculated. As the patient is not obese actual body weight can be used in the equation. From dosing guides allopurinol 100 mg daily is recommended. Discussion of Case 4 An man aged 70 complains of weakness and dizziness. He has been taking paroxetine 20 mg daily for about 4 weeks. There are no other recent medication changes. A screen of urea and electrolytes is performed. The only significant result is a serum sodium concentration of 121 mmol/L (hyponatraemia). The patient does not appear dehydrated or oedematous but because of the low sodium there is a suggestion to administer salt containing fluids. However, when urine sodium is checked it is high, indicating the possibility of SIADH (i.e. water retention, dilution of serum sodium and concentrated urine). This diagnosis is also supported by the time frame of presentation of the hyponatraemia as SSRI induced SIADH often manifests 4 – 6 weeks after the drug is started. Correct treatment is to stop the paroxetine, restrict fluid intake and monitor.

24