Clinical Lab Lec 4 PDF

**Clinical laboratory** Cardiac Markers =============== Cardiac markers are biomarkers measured to evaluate heart function. Most of the early markers identified were enzymes, and as a result, the term \"***cardiac enzymes***\" is sometimes used. However, not all of the markers currently used are e...

**Clinical laboratory** Cardiac Markers =============== Cardiac markers are biomarkers measured to evaluate heart function. Most of the early markers identified were enzymes, and as a result, the term \"***cardiac enzymes***\" is sometimes used. However, not all of the markers currently used are enzymes. Cardiac markers or cardiac enzymes are proteins that leak out of injured myocardial cells through their damaged cell membranes into the bloodstream. Until the 1980s, the enzymes SGOT and LDH were used to assess cardiac injury. Now, the markers most widely used in detection of MI are MB subtype of the enzyme creatine kinase and cardiac troponins T and I as they are more specific for myocardial injury. An ECG still remains the most specific diagnostic tool in evaluating the patient with chest pain however, the initial ECG may be negative/non-diagnostic in \> 40% of AMI cases the perfect cardiac marker test, with a 100% early sensitivity + 100% specificity in diagnosing an AMI, does not exist different cardiac marker tests are used in varying combinations. **Why it is done?** Cardiac enzymes levels help diagnose chest pain or other signs and symptoms of a heart attack. Limitations ----------- Depending on the marker, it can take between 2 to 24 hours for the level to increase in the blood. Additionally, determining the levels of cardiac markers in the laboratory takes time. Cardiac markers are therefore not useful in diagnosing a myocardial infarction in the acute phase. The clinical presentation and results from an ECG are more appropriate in the acute situation. Lactate Dehydrogenase (LDH) =========================== Lactate dehydrogenase (LDH, or LD) is an enzyme that is found in almost all body tissues but only a small amount of it is usually detectable in the blood. It usually stays contained within the tissues cells. When cells are damaged or destroyed, however, they release LDH into the bloodstream, causing blood levels to rise. For this reason, LDH is used as a general marker of injury to cells. Function -------- Lactate dehydrogenase catalyzes the interconversion of pyruvate and lactate with concomitant interconversion of NADH and NAD+. It converts pyruvate, the final product of glycolysis to lactate when oxygen is absent or in short supply. This reaction is known as anaerobic homolactic fermentation and is an important way to regenerate NAD+ to allow glycolysis to continue. **LDH isoenzymes** LDH functions as a tetramer and is made of two kinds of subunits, H and M, each of which are encoded by a different gene. This results in 5 different isoenzymes (2 homotetramers and 3 heterotetramers). The M subunit is found predominantly in anaerobic tissues including skeletal muscle and liver. The H subunit is more commonly found in tissues with a ready source of oxygen and that metabolize lactate including the heart and the brain. *LDH-1 (4H)* - in the heart *LDH-2 (3H1M*) - in the reticuloendothelial system *LDH-3 (2H2M*) - in the lungs *LDH-4 (1H3M*) - in the kidneys *LDH-5 (4M)* - in the liver and striated muscle Usually LDH-2 is the predominant form in the serum. A LDH-1 level higher than the LDH-2 level (a \"flipped pattern\"), suggests myocardial infarction. Sample ------ Serum sample is used Source of error Strenuous exercise can cause temporary elevations in LDH Haemolysis of blood can cause false positive result **Interpretation:** Elevated levels of LDH and changes in the ratio of the LDH isoenzymes usually indicate some type of tissue damage. Usually LDH levels will rise as the cellular destruction begins, peak after some time period, and then begin to fall. **Cardiac disease:** it can be used as a marker of myocardial infarction. Following a myocardial infarction, levels of LDH will rise within *24 to 48 hours*, peak at *3-4 days* and remain elevated for up to *10 days*. In this way, elevated levels of LDH can be useful for determining if a patient has had a myocardial infarction if they come to doctors several days after an episode of chest pain. LDH level is directly proportional to the infraction size. *[Note:]* *LDH level can be elevated in other cardiac disease such as Myocarditis and rheumatic fever* **Non-cardiac disease:** elevated levels of LDH may be seen in the following conditions: Hemolytic anemia and Pernicious anemia but it decreases with treatment. Liver disease: LDH increase in liver disease (mainly LD4 and LD5) but not as much as GOT and GPT. Toxic hepatitis and carcinoma of liver cause high elevation while Viral hepatitis and obstructive jaundice cause moderate elevation. Muscular dystrophy: It increase in patients with progressive muscle dystrophy especially in early and middle stage of the disease then it decreases with progression of the disease. Some cancers (especially lymphoma and leukemia) as cancer cells have a high rate of turnover with destroyed cells leading to an elevated LDH activity. **Colorimetric Method for the Estimation of Lactate Dehydrogenase** **Principle** This method is based on the reduction of pyruvate to lactate in the presence of NADH by the action of lactate dehydrogenase. LDH **Pyruvate + NADH +(H+) → Lactate + (NAD+)** The pyruvate that remains unchanged react with 2,4 dinitrophenyhydrazone, which is determined calorimetrically in an alkaline medium **Procedure:** Pipette into cuvette NADH 0.9 ml (place in water bath at 27 °C for 2\_3 min ). Sample 0.1 ml ( mix , incubate in water bath at 37 °C for exactly 30 min ). Colour reagent 1.0 ml (mix , allow to stand at room temperture for 20 min ). Sod. Hydroxide (1.6 % ) 10 ml ( mix, allow to stand for 10 min , read absorbance of sample against D.W ). Creatine kinase =============== Overview -------- Creatine kinase (CK), also known as creatine phosphokinase (CPK) or phosphocreatine kinase, is an enzyme expressed by various tissues and cell types. **What is Creatine?** Creatine is produced by the body at rate of 1-2g/day from the amino acids, glycine, arginine and methionine. The liver is the major site of production but some is also produced in the kidney and pancreas. It exists as free creatine and creatine phosphate (CP). Function -------- CK catalyses the conversion of creatine and consumes adenosine triphosphate (ATP) to create phosphocreatine and adenosine diphosphate (ADP). This reaction is reversible, such that also ATP can be generated from PCr and ADP. Role of phosphocreatine (PC) ---------------------------- In tissues and cells that consume ATP rapidly, especially skeletal muscle, phosphocreatine serves as an energy reservoir for the rapid buffering and regeneration of ATP in situ. Thus when muscle contracts, ATP is consumed to form ADP, in this case CK catalyse the rephosphorylation of ADP to form ATP using Crp as the phosphorylation reservoir. CK isoenzymes ============= In the cells, the CK enzymes consists of two subunits, which can be either B (brain type) or M (muscle type). There are, therefore, three different isoenzymes: CK-MM (CK3), CK-BB (CK1) and CK-MB (CK2). **CK-BB** occurs mainly in brain tissues, and its levels do rarely have any significance in bloodstream. Skeletal muscle expresses **CK-MM** (98%) and low levels of **CK-MB** (1%). The myocardium (heart muscle), in contrast, expresses **CK-MM** at 70% and **CKMB** at 25--30%. **Sample:** Serum sample is used **Interpretation:** [Cardiac disease:] CK total is elevated rapidly after MI In the first ***4 to 6 hours*** after a heart attack, the concentration of CK in blood begins to rise. It reaches its highest level in ***18 to 24 hours*** and returns to normal within ***2 to 3 days***. CK level is directly proportional to the infraction [Muscular disease:] Because most of the CPK in the body normally exists in muscle, a rise in the amount of CPK in the blood indicates that muscle damage has occurred. **Physiological rise:** sever muscular exercise, cramps, repeated muscular injections cause transient CK rise for 2-4 days. **Pathological rise:** - Parasitic infection caused by trichinosis - Convulsions and muscle spasms - Duchenne\'s muscular dystrophy Caused by mutation in the gene of dystrophin protein. In this case high plasma activities are found from birth and before the onset of clinical signs During the early clinical stages at the disease, very high activity usually present but at terminal stage of the disease, the level tend to be fall when the mass of functioning muscle diminish with progression of the disease Creatine kinase-MB (CK-MB) ========================== Serum CK-MB mainly comes from myocardial tissue so it is the first cardiac enzyme to be elevated after MI In the first ***2 to 4 hours*** after a heart attack, the concentration of CK-MB in blood begins to rise. It reaches its highest level in ***12 to 24 hours*** and returns to normal within ***1 to 3 days***. The sensitivity at 4 hours is \< 50%, but the sensitivity should reach \~ 100% for AMI 10 -- 12 hours after the onset of the chest pain an AMI cannot be ruled-out before \~ 9 - 10 hours after the onset of symptoms; longer if the patient has ongoing chest pain. **Why CK-MB is recommended?** Used when very early conformation of diagnosis is required within 4 h of the infarction When an increased CK total is suspected to be due to release of enzyme from skeletal muscle e.g. after intramuscular injections In patient suspected to having had a second infarction with few days and the first where the second peak rise of CK-MB is noted Small females with a small total body muscle mass may have low serum CK levels, which may not rise above the threshold value after an AMI the more specific CK-MB test is recommended in these cases. Angina pectoris =============== Commonly known as angina, is severe chest pain due to ischemia (a lack of blood and hence oxygen supply) of the heart muscle, generally due to obstruction or spasm of the coronary arteries (the heart\'s blood vessels). **Causes:** Coronary artery disease, the main cause of angina, is due to atherosclerosis of the cardiac arteries. Myocardial ischemia comes about when the myocardia (the heart muscles) receive insufficient blood and oxygen to function normally because of decreased supply to the myocardia. This inadequate perfusion of blood and the resulting reduced delivery of oxygen and nutrients is directly correlated to blocked or narrowed blood vessels. There is a weak relationship between severity of pain and degree of oxygen deprivation in the heart muscle (i.e., there can be severe pain with little or no risk of a heart attack, and a heart attack can occur without pain). Myocardial infarction (MI) ========================== Myocardial infarction (MI) or acute myocardial infarction (AMI), commonly known as a heart attack, is the interruption of blood supply to part of the heart, causing heart cells to die (necrosis). This is most commonly due to occlusion (blockage) of a coronary artery Signs and symptoms ------------------ Classical symptoms of acute myocardial infarction include sudden chest pain (typically radiating to the left arm or left side of the neck). Shortness of breath (dyspnea) occurs when the damage to the heart limits the output of the left ventricle, causing left ventricular failure and consequent pulmonary edema. Nausea, vomiting and palpitations: These symptoms are likely induced by a massive surge of catecholamines from the sympathetic nervous system which occurs in response to pain. Excessive form of sweating, Anxiety, Loss of consciousness (due to inadequate cerebral perfusion and cardiogenic shock) and sudden death. *[Notes:]* Approximately one fourth of all myocardial infarctions are silent, without chest pain or other symptoms. These cases can be discovered later on electrocardiograms, or using blood enzyme tests. A silent course is more common in the elderly, in patients with diabetes mellitus. Creatine Kinase Activity Measurement (Kinetic Method) ===================================================== Principle: Kinetic determination of creatinine kinase:  The catalytic activity of CPK is determined by the measurement of NADPH formation. Procedure: Pipette into cuvettes: Working solution 1000 microLiter Then add 40 microliter of sample for 5 min. at 37 Mix and incubate at 37 for 2 min. read the initial absorbance at 365 nm and started timer immediately. Read again at constant intervals for 3min. *[Calculation:]* Calculate the average value of variations of absorbance per min OD\\min then apply the following formula: *[Reagents:]* Reagent 1(Imidazol acetate buffer PH 6.5, Magnesium acetate) Reagent 2 (substrate) D-glucose, Creatine phosphate, EDTA, Nacetyl cysteine, ADP, AMP Diadenosine pentaphosphate, NADP, Hexokinase (HK), G-6-PDH Troponins ========= The troponin complex consists of three distinct polypeptide components: troponin C (the calcium binding element), troponin-I (the actinomyosin ATPase inhibitory element; cTnI), and troponin-T (the tropomyosin binding element; cTnT). The troponin complex (cTnICT) serves to regulate the calcium-dependent interaction of myosin and actin and thus plays an integral role in muscle contraction. **Principles of Analysis:** a number of assays for troponin are commercially available. A variety of quantitative and semiquantitative point-of-care methods have also been developed. Current assays for cTnT and cTnI are two- or threesite immunoassays. All the assays are of the capture type, where an immobilized antibody specifically binds the troponin present in the serum or plasma. The captured troponin is then reacted with a second antibody, and in some assays, a third antibody that is coupled to an indicator molecule. The assays vary from each other by the types of antibody used, by the epitopes to which they bind, and by the type of indicator molecule that is used. Specimen -------- *[Sample Type]:* if serum samples are used, blood should be allowed to clot. Care should be taken when preparing specimens for testing from patients who have received anticoagulant therapy. These specimens may require additional time to clot. If plasma samples are used, blood is usually collected in a tube containing heparin anticoagulant. EDTA can split Ca2+-dependent cTn complexes and decrease troponin concentration in assays that measure preferentially these molecular forms. Either serum or heparinized plasma may be used as the sample type for most commercially available assays; whole blood is used for some pointof-care. *[Methods:]* however, several studies report significant differences in cTnI measured in serum and plasma, with plasma results reportedly being up to approximately 30% lower compared with serum. This tendency for lower results in plasma can result in failure to detect an early or small AMI. *[Interpretation]*: the evaluation of troponin concentration is a useful tool in the diagnosis of AMI. Cardiac troponin is released into blood within 2 to 4 hours of the onset of symptoms of AMI, peaks at 12 to 18 hours, and remains elevated for 5 to 14 days post infarction. The diagnostic sensitivity and specificity of cTnI and cTnT for acute coronary syndrome (ACS) is shown by the high specificity of troponins and the increasing sensitivity with time post-ACS. \*\*\* Good Luck \*\*\*