Introduction to Haematology and Anaemia - THEP2, 2nd Class PDF

Royal College of Surgeons in Ireland Medical University of Bahrain FUN-28: Clinical Enzymology and concepts of clinical testing Salim Fredericks FUN-28: Clinical Enzymology Learning objectives Explain the use of biochemical investigations for diagnosis and monitoring disease progression Des...

Royal College of Surgeons in Ireland Medical University of Bahrain FUN-28: Clinical Enzymology and concepts of clinical testing Salim Fredericks FUN-28: Clinical Enzymology Learning objectives Explain the use of biochemical investigations for diagnosis and monitoring disease progression Describe the general principles of enzyme measurement Define and classify enzymes and isoenzymes Discuss biomarkers of myocardial infarction Discuss biochemical investigations of liver disease Outline commonly used biochemical techniques to measure analytes in body fluids Describe the concepts of patient management and investigation of disease Patient management and clinical biochemistry Diagnosis Treatment Surgery or Medical Monitor outcome Disease and treatment Gaw et al Clinical Biochemistry: an illustrated colour text Use of clinical chemistry Principle uses of biochemical tests Screening Diagnosis Detection of sub clinical Confirmation or rejection disease of clinical diagnosis (e.g. PKU) (e.g. cTnT and cTnI) Monitoring Prognosis Natural history of Information regarding response to treatment likely outcome of disease (e.g. TDM or glucose in (e.g. serum cholesterol in diabetes) CAD) Cellular damage resulting from disease or trauma If it should be in a cell, it should stay in the cell: not in plasma Most analytes (proteins, enzymes or other molecules) used in diagnosis depend on the very high concentration of that substance within the cell relative to that in plasma. Enzymes and cell dysfunction Duchenne muscular dystrophy In patients with Duchenne muscular dystrophy there is a continual cycle of growth and rupture of myocyte of the skeletal muscle This abnormal production of muscle mass leads to a leaking of the content of the myocytes through cell rupture The contents of the myocyte’s cytoplasm ends up in the blood. This includes the enzyme CK Creatine kinase (CK) CK catalyses the reaction of creatine phosphate to creatine CK and the diagnosis of Duchenne muscular dystrophy Creatine kinase (CK) CK is an enzyme involved in energy production found in certain cells. These cells have unusually high energy demands CK is present in large amounts in skeletal muscle and cardiac muscle CK is found in the blood as a result of leakage from routine turn over of skeletal muscle Duchenne muscular dystrophy Patient should have elevated plasma activities of CK. This can be more than 10 x higher than reference range Asymptomatic female carriers of the DMD gene often (75%) have elevated plasma CK activities Muscle damage Excessive physical exercise Marathon runners New army recruits Surgery Skeletal muscle trauma Drug induced (cocaine)  General principles of enzyme measurement Cellular damage – why enzymes? Why measure enzymes? Relatively easy to measure compared with other proteins (technically and economically) Detection Enzymes as catalysts Increase the rate of a chemical reaction without being consumed A small amount of enzyme can convert a much larger amount of substrate enzyme Substrate Product (cofactors) The appearance of a small amount of enzyme in the bloodstream from damaged tissue can be detected with great sensitivity Coupled reaction Lactate NAD+ Reduced Oxidised Lactate Phenazine Tetrazolium dehydrogenase Methosulphate salt Pyruvate NADH Oxidised Reduced (coloured) Colour change measured Ingredients: Lactate NAD+ Phen. Meth (oxidised) Tetrazolium salt (oxidised) Sample Also - enzyme-coupled assays  Assay of Creatine kinase (CK) Creatine kinase In vitro CK Creatine phosphate Creatine reaction ADP ATP NADPH production is Glucose-6- Glucose phosphate Hexokinase monitored by NADP the Glucose-6- absorbance phosphate DH change at 340 NADPH + H (fluorescent) + nm 6-Phosphogluconate Myocardial infarction Infarction : death of part or the whole of an organ that occurs when the artery carrying its blood supply is obstructed by a blood clot Infarct: small localised area of dead tissue thus produced Plasma enzyme activities following MI Total creatine kinase The “total CK” assay measures all CK activity present in a plasma specimen. However there are several molecular forms of the enzyme CK. The assay described measures total CK activity  What are enzymes and isoenzymes? Isoenzymes Enzymes are classified by the reactions they catalyze - substrate-ase Isoenzymes are different molecules that catalyze the same reaction Multiple gene loci Related to division of function between and within different cell types and tissues Not uniform throughout the body Tissue-specific – certain genes may be expressed exclusively in one tissue The presence of a specific isoenzyme in the plasma may indicate the damage to a specific tissue Cellular localization within specific organelles Quaternary structure of enzymes/isoenzymes Different isoenzymes can be formed from different combinations of subunits Eg Hexokinase (monomer) Eg Creatine Kinase (dimer) Eg Lactate Dehydrogenase (tetramer) Tietz 9-2 Isoenzymes Electrophoresis – Isoenzymes may have different electrophoretic mobility, will give characteristic pattern on electrophoresis – Detection by chromogenic substrate – - detects active enzyme only – Immunological detection – - detects active and inactive enzyme – (Densitometry) Differential activity – - Isoenzymes can have differing activities towards different substrates – - can have different activities under different reaction conditions Other properties Resistance to inactivation, solubility, etc CK isoenzyme plasma changes following MI MM MM MB MB Before After Myocardial Infarction Creatine kinase isoenzymes Tissue CK activity CK3 (MM) CK2 (MB)CK1 (BB) (U/g Wet Wt) (%) (%) (%) Skeletal muscle 2500 98.9 1.1 0.9 Heart 473 78.7 20 1.3 Brain 555 0 2.7 91.3 GI tract 176 2.2 0 97.8 Prostate 114 6 0 100 Liver ~1 0 0 100 Uterus 115 2.3 0 97.4 Total CK is potentially used to assess skeletal muscle damage and cardiac damage. CK-MB good for cardiac, CK-MM good for skeletal muscle.  Biochemical diagnosis of AMI How good are the clinical chemists compared to the cardiologists? Clinical Biochemistry: An Illustrated Colour Text, A. Gaw, M.J. Murphy, R.A. Cowan, J. O’Reilly, M.J. Stewart, J. Shepard Which enzyme test is used for which condition? Plasma enzyme levels Dependant on: Rate of release from damaged cells; Rate of damage to cells; Extent of cell damage; In the absence of cell damage, rate of release depends on Rate of cell proliferation (malignancy) Degree of induction of enzyme synthesis Rate of clearance (removal) from circulation Distribution of clinically important enzymes Enzyme Principal source Principal clinical application Acid phosphatase Prostate, erythrocyte (prostate cancer) (PSA used more now) Alanine aminotransferase Liver, skeletal/cardiac muscle Hepatic parenchymal disease Alkaline phosphatase Liver, bone, intestinal mucosa, Bone & hepatobiliary disease placenta, kidney Amylase Salivary glands, pancreas Pancreatic disease Aspartate Liver, skeletal/cardiac muscle, kidney, hepatic parenchymal (Myocardial aminotransferase erythrocyte infarction & muscle disease) Cholinesterase Liver Organophosphorus insecticide poisoning, suxamethonium sensitivity Creatine kinase Skeletal/cardiac/smooth muscle, brain Myocardial infarction, muscle disease Glutamate Liver Hepatic parenchymal disease dehydrogenase γ glutamyltransferase Liver, kidney Hepatobiliary disease Lactate dehydrogenase Heart, liver skeletal muscle, Haemolysis, myocardial infarction, erythrocytes platelets hepatic parenchymal disease Trypsin(ogen) Pancreas Pancreatic disease Selection of enzyme tests Distribution of enzyme among various tissues – Tissue / plasma concentration gradient – Intracellular localisation Convenience of enzyme assay – Knowledge of plasma enzyme characteristics Half-life in blood Mode of clearance Liver enzymes Liver enzymes Plasma enzymes used to assess hepatic function include: AST and ALT - aspartate and alanine aminotransferase (formally called transaminases and still abbreviated to AST and ALT respectively) ALP - alkaline phosphatase GGT - -glutamyl transferase Liver function –Metabolism Glycogen synthesis and breakdown (glycogenolysis) Gluconeogenesis Fatty acid metabolism –Synthesis Plasma proteins including albumin Coagulation factors Lipoproteins Bile acids –Excretion and detoxification Bilirubin Amino acids and NH3 Cholesterol and steroid hormones Drugs, Toxins Liver Disease Pathological changes – Liver cell damage: acute hepatitis, chronic hepatitis, toxin action, alcohol abuse – Cholestasis: gallstones, cancer at the head of the pancreas – Reduced mass of functioning cells (e.g cirrhosis, terminal hepatic failure) – Infiltrative disorders - liver is invaded or replaced by nonhepatic substances such as neoplasm or amyloid. Tests for liver disease - (“Liver function” tests) – Liver cell damage - enzymes - ALT, AST – Synthetic function - prothrombin time, albumin – Conjugating capacity - conjugated bilirubin – Cholestasis - enzymes - Alk phos, γ-GT (also serum bilirubin) Hepatocellular damage Aspartate and Alanine Transaminase measurements – Abbreviated: » AST (aspartate transaminase) » ALT (alanine transaminase) – Also called ‘aminotransferase’ ALT more liver-specific than AST (Both enzymes are widely distributed in body tissues, but ALT is present in only small amounts except in the liver) AST has cytoplasmic and mitochondrial isoenzymes, tends to be released more than ALT in chronic hepatocellular disease Choleostasis failure of normal amounts of bile to reach the intestine Causes: mechanical block (gallstone, tumour); liver disease; drug induced, e.g. phenobarbitol Alkaline phosphatase (ALP) and -glutamyl transferase (-GT) – normally “anchored” to membranes of hepatocytes, released in only small amounts in hepatocellular damage. In choleostasis, synthesis of these enzymes is induced and they are made soluble (high concs of bile acids)  GT more liver-specific – ALP also raised in bone disease Changes in -GT and ALP often parallel each other in choleostatic disease  Causes of an increased plasma AST acute hepatitis and liver Often > 10 x ULN (levels necrosis may sometimes exceed major crush injuries 100 x ULN in these severe tissue hypoxaemia conditions) myocardial infarction 5-10 x ULN following surgery or trauma skeletal muscle disease cholestasis chronic hepatitis physiological (neonates) usually ALT disease is due to Raised γ-GT excess alcohol intake Increased mean cell volume (MCV) Hyperbilirubinaemia Raised Alkaline Phosphatase Increased prothrombin time (INR) Hypertriglyceridaemia Decreased K+, PO43-, Mg2+  Commonly used biochemical techniques to measure analytes in body fluids Common methods There are many biochemistry methods and techniques. In practice only a few are used in clinical chemistry  Spectrophotometric  Other photometric techniques Turbidimetry, fluorometry, Chemiluminescence  Immunoassay  Chromatographic  Components of a spectrophotometer A, exciter lamp B, entrance slit C,monochromator D, exit slit E, cuvette F, photodetector G, LED display Beer Lambert Law: Absorbance =  x C x L Common clinical chemistry assays Reduction NAD produces a new, broad absorption band with a max at 340 nm. The production of NADH during an enzyme- catalyzed oxidation can be conveniently followed by observing the appearance of the absorbance at 340 nm. Immunoassays With modern computing power, robotics and improved fluorescence and chemiluminescence detection the number of types of immunoassays and phenomenally high.  Diagnostic companies are competing to patent new technologies  The area has become filled with ‘black box’ technologies and methods Chromatography Principle of thin-layer chromatography (TLC). Two solutes, A and B, are applied to the polar silicate strip. A is more polar than B and has a higher affinity, therefore, for the polar stationary phase than for the nonpolar mobile phase (usually methanol in chloroform). This relative affinity of A is, moreover, higher than the affinity of B for the polar phase. Therefore, A separates out first on the strip, and B migrates further on the strip. LC; Liquid Chromatography (HPLC) Background reading Clinical Chemistry (5th Ed); – W.J. Marshall and S.K. Bangert Clinical Biochemistry(6th Edn); Smith, Beckett, Walker and Rae Clinical Chemistry in the Diagnostics and Treatment. J. F. Zilva, P. R. Pannall and P. D. Mayne McPherson & Pincus: Henry's Clinical Diagnosis and Management by Laboratory Methods, 21st ed Tietz “fundamentals of clinical chemistry” - Ch. 9 Bishop et al “Clinical Chemistry” esp. ch 10, 22, 23, 25