Clinical Laboratory Medicine

Clinical Laboratory Medicine mgr inż. Agnieszka Kamińska Bibliography Clinical Laboratory Medicine edited by: Richard C. Tilton, Albert Balows et all, 1992.  Clinical Guide to Laboratory Test edited by: Norbert W. Tietz, Third Edition.  Laboratory organization  Core Laboratory (73% of volume) – Chemistry, hematology, toxicology, immunochemistry, special chemistry, coagulation, urinalysis   Microbiology (16 % of volume) – Blood/urine cultures, serology Transfusion Services (3% of volume) – Blood typing and cross-matching Chemistry       Electrolytes, glu, BUN, creat, phos, TP, Bili, Mg, Ca, cholesterol, triglycerides, etc. Blood gases: pO2, pCO2, pH, calc. Parameters Immunochemistry: endocrine, specific protein, tumor markers Toxicology: DAU, TDM Urinalysis Special Chemistry: electrophoresis, L/S ratio, FLM, osmometry Reasons for POC testing  Tests are of urgent importance, and results will affect the immediate management of the patient – Blood gases, electrolytes  Tests are so common, simple and cheap that it is more economical to perform them at the point of care – Blood glucose, urinalysis Plasma/Serum differences   Plasma concentration greater than serum: – Ca (+0.9%), LD (+2.7%), TP (+4.0%) Plasma concentration less than serum: – Alb (-1.3%), ALKP (-1.6%), HCO3- (-1.8%), CK (-2.1%), PO4= (-7.0%), K+ (-8.4%) Prolonged venous stasis Increases TP, Fe, cholesterol, AST, bilirubin  Decreases potassium  Supine vs. sitting or standing  The following may decrease by 5-15% in the supine patient: – – – – – – – – Total protein Albumin Lipids Iron Calcium Enzymes Ig Thyroxine Significantly affected by hemolysis:  Total protein, albumin, lipids, iron, calcium, enzymes, bilirubn, cholesterol, triglycerides, norepinephrine, renin, aldosterone, potassium, magnesium, phosphorous Exertion-related changes  Recent strenuous exercise increases: – Acid phosphatase, ALT, AST, creatinine, phosphorous, CK  Recent strenuous exercise decreases: – Iron, lipids, potassium Other factors affecting lab results    Diurnal variations – Cortisol, iron, estriol, glucose, catecholamines Age – Creatinine, BUN, ALKP, drug metabolism Smoking – Ammonia, CO-Hb Profile - a variety of test; can encompass a large spectrum of assays for a general overall picture off health or might concentrate on particular tissue or an area of special interest Profiles 1. 2. 3. 4. 5. 6. 7. 8. Renal profile Liver profile Bone profile Heart profile Lipid profile Thyroid profile Pancreatic profile Control profile Five primary function of the kidney      Removal of waste products (nitrogenous waste, acids, etc.) Retention of nutrients (electrolytes, protein, water, glucose) Acid-base balance Water and electrolyte balance Hormone synthesis (erythropoietin, renin, vitamin D) Removal of waste products The removal of body waste is important since most of these substances are toxic to the body at the elevated levels The wastes removed from the body by the kidney include acids derived from the body’s general metabolic process and nitrogenous wastes. The nitrogenous wastes include: 1. Urea - which is the end-product of catabolism amino acids and proteins. Urea is formed by the urea cycle in the liver. 2. Creatinine – which is formed by spontaneous hydrolysis and cyclization of muscle creatine. Creatine serves as the primary energy reservoir of muscle cells by forming creatinephosphate. 3. Uric acid - which is the end-product metabolite of the nitrogenous purine bases, adenine and guanine, used for DNA and RNA synthesis. Nutrient conservation Just as it is important for body to rid itself of the waste product of metabolism, it must also conserve important body nutrients and other important substances. These nutrients, which are important because they help maintain body function, include: protein and amino acids, glucose, electrolytes especially sodium, calcium, chloride and bicarbonate, water Acid-base,water and electrolyte balance Intimately linked with its excretion and conservation function, is the kidney’s role of maintaining the pH of the body’s fluids. The kidney achieves this by eliminating mineral acids and conserving alkali as needed. Similarly, the excretion and retention of salts and water is the basis of the kidney’s role in maintaining overall water and electrolyte balance Hormone synthesis The kidney also serves as the site of synthesis of three endocrine hormones : 1. erythropoietin, 2. renin, 3. vitamin D The kidneys as regulatory organs “The kidney presents in the highest degree the phenomenon of sensibility, the power of reacting to various stimuli in a direction which is appropriate for the survival of the organism; a power of adaptation which almost gives one the idea that its component parts must be endowed with intelligence.” E. Starling (1909) Review of Renal Anatomy and Physiology  The kidneys are a pair of fist-sized organs that are located on either side of the spinal column just behind the lower abdomen (L1-3). A kidney consists of an outer layer (renal cortex) and an inner region (renal medulla).   The functional unit of the kidney is the nephron; each kidney has approximately 106 nephrons. Renal anatomy Cortex Pelvis Capsule Medulla To the bladder The Nephron Afferent arteriole Glomerulus Bowman’s capsule Proximal tubule Distal tubule Collecting duct Renal artery Henle’s Loop What gets filtered in the glomerulus?  Freely filtered  Some filtered  None filtered – H2O – Immunoglobul – 2+, K+, Cl-, – Na microglobulin ins HCO3-, Ca++, Mg+, PO4, etc. – RBP – Ferritin – Glucose – Cells – 1– Urea microglobulin – Creatinine – Albumin – Insulin Then what happens?  If 200 liters of filtrate enter the nephrons each day, but only 1-2 liters of urine result, then obviously most of the filtrate (99+ %) is reabsorbed. Reabsorption can be active or passive, and occurs in virtually all segments of the nephron.  Glomerular filtration Vascular space Glomerlular capillary membrane Mean capillary blood pressure = 50 mm Hg Bowman’s space  2,0 Liters per day (25% of cardiac output) BC pressure = 10 mm Hg  200 Liters per day Onc. pressure = 30 mm Hg Net hydrostatic = 10 mm Hg GFR  130 mL/min Reabsorption from glomerular filtrate % Reabsorbed Water Sodium Potassium Chloride Bicarbonate Glucose Albumin Urea Creatinine 99.2 99.6 92.9 99.5 99.9 100 95-99 50-60 0 (or negative) How does water get reabsorbed?  Reabsorption of water is passive, in response to osmotic gradients and renal tubular permeability. – The osmotic gradient is generated primarily by active sodium transport – The permeability of renal tubules is under the control of the renin-angiotensin-aldosterone system.  The driving force for water reabsorption, the osmotic gradient, is generated by the Loop of Henle. Regulation of distal tubule Na+ permeability JGA  Na+  BP Renin Angiotensinogen Angiotensin I Angiotensin II Angiotensin III Aldosterone Na+ vasoconstriction Adrenal cortex Na+ Regulation of H2O reabsorption Pituitary Plasma hyperosmolality ADH (vasopressin) H2O H2O Renal Medulla (osmolality 1200 mOsm/Kg) Summary of renal physiology TRPF (Filtered and secreted) Filtration - Reabsorption + Secretion = Elimination GFR (Filtered but not reabsorbed or secreted) Renal Profile Na K  Urea  Creatinin  Urea acid  Basic diagnostics Blood – morphology, creatinine, creatinine clearance, electrolytes (Na, K, Cl), phosphate, osmolality, protein.  Urine (24 h urine collection) - protein, albumin.  Na and K     The main function is the sodium/potasium pump, which is under the hormonal control of aldosterone. Aldosterone stimulates renal reabsorption of Na and excretion of K+. The increase in the plasma concentration of sodium and water results in an expanded plasma volume and increased blood pressure. Na and K can be accurately and precisiely measured by ion-selective electrodes (ISE). Plasma or serum is approximately 93,5% water, 5,4% protein, 0,6% lipid and 0,9% other molecules. Sodium and potasium ion are present in the water fraction. Na and K references range Na (mmol/L) Male K (mmol/L) 3,5 - 4,5 3,4 - 4,4 136 - 145 Female Na and K    Hypo- and hypernatremia can be caused by a number of clinical conditions such as renal disease, liver disease, heart failure, diabetes, inapproprite (iatrogenic) treatment with fluids and electrolytes, gastrointestinal diseases, burns and aderenal insufficiency. Hypokalemia – treatment with diuretics without sufficient supplementation with potassium. Prolonged treatment with corticosteroids. Hyperkalemia – acute or chronic renal failure.As in the case of sodium clinical conditions associated with hypoor hyperkalemia may be associated with increased or decreased level of total body potassium. Clinical conditions associated with hyperkalemia include acidosis crush injuries, hypoxia, renal failure, while hypokalemia may be associated with alkalosis diuretic therapy and increased potassium loss from renal or gastrointestinal diseases. Creatinine    Creatinine – which is formed by spontaneous hydrolysis and cyclization of muscle creatine. Creatine serves as the primary energy reservoir of muscle cells by forming creatine phosphate. Serum and plasma - material to analysis Indication: acute and chronic renal failure (ARF, CRF), diabetes, glomerular dysfunction Method of analysis: Jaffe method-end-point Jaffe method-kinetic Enzymatic method  Creatinine - reference ranges mg/dL Child <10 y Adult male Adult female 0,04 - 0,59 0,62 – 1,10 0,45 – 0,75 mol/L 4 - 52 55 - 96 40 - 66 mg/dl x 88,4 = mol/l Creatinine  High range: 1. Regular kidney function: dehydration, acromegaly, 2. Acute renal failure: -prerenal causes: hypovolemia, anaphylactic shock, septic shock, cardiogenic shock; -renal causes: acute tubular necrosis from toxins, contrast medium, hemolysis, myeloma, septicemia, eclampsia of pregnancy, glomerular nephritis, systemic diseases; - postrenal causes: retention of urine, narcotic, parasympathetic drugs, rupture of bladder 3. Chronic renal failure: interstitial renal inflammation, diabetic nephropathy, arterial hypertension, connective tissue diseases, polycystic kidney. Creatinine clearance   Serum, plasma and 24 h collected urine - material to analysis Indication: therapy of nephrotoxical drugs, diabetes, hypertension, hyperuricemia connective tissue diseases acromegaly Creatinine clearance The formula for circulating CL (Creatinine clearance) : CL(ml/min) = U cr (mg/dl) x V(ml) P cr (mg/ml) x t (min) Where: U cr-creatinine concentration in urine P cr- creatinine concentration in plasma or serum V- volume of urine t -time of collection urine The 24-hour creatinine clearances can be normalise to a body surface area of 1,73m²: CL (ml/min) = (CL x 1,73)/A Where: A – surface area in square meters Creatinine clearance-reference ranges in ml/min/1,73 m² surface area Child 0-1 y Child 1-14 y Adult 20-29 y Adult 30-39 y 72 45 - 109 Adult 40 < Male Female 94 - 140 72 - 110 59 - 137 71 - 121 for each decade thereafter, values decrease  6,5 ml/min/1,73m² 1. 2. Lower values are indicative of renal dysfunction High values are indicative of pregnancy or early diabetes Urea 1. 2. 3. 4. 5. 6. Urea - which is the end-product of catabolism amino acids and proteins. Urea is formed by the urea cycle in the liver. Serum and plasma – material to analysis Method of analysis: GLDH (glutamate dehydrogenase) reaction Berthelot reaction References range: 10-50 mg/dl (plasma,serum) Volume of urea depends on protein amount in diet High volume – tissue disintegration (bleeding from digestive tract), huge protein amount in diet, dehydration, renal failure. Uric acid 1. 2. 3. 4. Uric acid - which is the end-product metabolite of the nitrogenous purine bases, adenine and guanine, used for DNA and RNA synthesis. Serum, heparinized plasma and 24 h collected urine - material to analysis. Indication: - serum: primary and secondary gout, nephrolithiasis, lymphoma, leukemia, anemia, renal diseases, overweight, psoriasis; -urine: hyperurykemia. Method of analysis: phosphotungsic acid spectrophotometric(non-specific), uricaseenzymatic (colometric and differential absorption) Uric acid  Reference ranges Child <12 y Adult - male Adult - female Common diet Purin-free diet Serum 2,0 – 5,5 mg/dl 4,4 – 7,6 mg/dl 2,3 – 6,6 mg/dl Urine 250 – 750 mg/24h Male: < 420 mg/24h Female: slightly lower Uric acid     High level in serum: primary and secondary gout, Lesch- Nyhan disease, cytostatics treatment, malignant neoplasma, renal dysfunction, hyperparathyroidism, hyperthyroidism, alcoholism. High level in urine: primary gout Low level in serum: xantinuria, overdosage allopurinol, tubular diseases, liver failure, uretreolithiasis. Low level in urine: liver failure, ketoacidisis and lactic acidosis, hyperparathyroidism, hyperthyroidism, saturnism, bromism Liver       is the largest gland in the body, is also the principal organ for metabolism of carbohydrates, lipids, porphyrin, bile acids and proteins is a major storage site for iron and other metals, glycogen, lipids and vitamins and play a central role in nitrogen metabolism, is involved with the metabolic interconversions of amino acids and synthesis of nonessential amino acids, is also largely responsible for the production of metabolic end products such as creatinine, urea, ammonia and uric acid, which can be more easily excreted, is the major organ involve with the detoxification of exogenous organic materials ingested by man. Liver profile          Bilirubin Albumin ALT AST GGTP ALP NH3 Urea Uric Acid Bilirubin The breakdown of heme-containing proteins, primarily hemoglobin, result in the production of about 250 mg of bilirubin per day.This occurs in the spleen as a result of hydrolysis of heme to release the iron and form the intermediate biliverdin, which is reduced to bilirubin. The iron is bound to transferrin and is reutilized for hem synthesis. The bilirubin is bound to albumin and is transported to the liver. Once the bilirubin is transported to the liver it is released from albumin and react with one to two molecules of glucuronic acid. This increase the water solubility of bilirubin so that it can be excreted in the bile. Bilirubin metabolism blood (hemolysis) Free hemoglobin binding by haptoglobin werdoglobin kidneys Globin, Fe biliverdin Reticulumendothelial system Unconjugated bilirubin binding by albumin React with glucuronic acid liver Entheropathic circulation Conjugated bilirubin Urobilinogen intestine Urobilin, serkobilin Bilirubin 1. Serum and heparinized plasma – material to analysis. 2. Method of analysis: Diazo reaction, enzymatic reaction. 3. Indication: diagnosis and control course of jaundice 4. Bilirubin fraction count: delta bilirubin = total bilirubin - unconjugated bilirubin conjugated bilirubin new-borns bilirubin = unconjugated bilirubin conjugated bilirubin Bilirubin - reference ranges total bilirubin [mg/dl] unconjugate conjugate d bilirubin d bilirubin [mg/dl] [mg/dl] Delta bilirubin [mg/dl] Adult New-borns <1,1 <13 <1,1 <11 <0,3 <0,6 <0,2 <0,2 Bilirubin Increase total bilirubin: Hemolysis, hemolytic jaundice, Crigler-Najjar syndrome. Increase conjugated bilirubin: Hepatitis, toxic jaundice, drug poisoning, mushroom poisoning, Dubin-Johns disease, Wilson disease, galactosemia, mucoviscidosis, hepatic cirrhosis, cholecystlithiasis, pancreas tumour Physiological changes: Increase during pregnancy and new-borns Enzymes 1. Use internationl unit of enzymes activity IU IU = 1 micromole/minute or Katal = 1 mole/sec 1IU = 1,67 nK 2. Isoenzymes (isozymes) – the multiple natural forms of an enzyme catalyzing the same reaction in a single species. Isoenzymes are to be distinguished on the basis of electrophoretic mobility closest to the anode (+) ALT - SGPT 1. 2. 3. Enzyme – alanine aminotransferase. Occurs in plenty specially in cytoplasm parenchymal liver cells and wall’s tubular cells. Less amount of ALT is in cardiac muscle, a small amount of ALT occures in several (all) tissues. ALT is organ-unspecific enzyme. 4. 5. Indication: liver diseases and cholepathy Serum and heparinized plasma – material to analysis. 6. Method of analysis: kinetic method (optic test) ALT- reference ranges Male Children <1 y Children and adult 1- 60 y Adult 60-90 y Adult > 90 y Female 13-45 U/L 10 – 40 U/L 7 - 35 U/L 13 - 40 U/L 6 – 38 U/L 10 – 28 U/L 5 – 24 U/L ALT - SGPT Increase activivty:  - acute virus hepatitis, toxic injury,  - hepatic cirrhosis, chronic active hepatitis, chronic persistent hepatitis, cholestasis, severe shock, right heart failure, left heart failure,  -fatty liver, primary liver tumour, drug-induced liver demage, metastases to liver, cholangitis, severe burns, chronic alcohol abuse AST - SGOT 1. 2. Aspartate aminotransferase Occurs in mitochondria (70%) and cytoplasm (30%) hepatocytes and muscle cells, renal tubule cells, erythrocytes, Indication: liver diseases, cholepathy, acute myocardoal infarction (supplementary examination), sceleton muscle disease. Serum and heparinized plasma – material to analysis. Method of analysis: kinetic method (optic test) de Ritis index – AST/ALT 3. 4. 5. 6. AST- reference ranges Male Newborn 0-10 day Children 10 day- 1y Children and adult 1-60 y Adult 60-90 y Female 47 – 150 U/L 9 – 80 U/L 15 – 40 U/L 19 - 48 U/L 13 – 35 U/L 9 - 36 U/L Adult > 90 y 11 - 38 U/L 18 - 30 U/L AST - SGOT Increase activivty:  - acute virus hepatitis, toxic liver injury, progressive muscular dystrophy.  - hepatic cirrhosis, chronic active hepatitis, chronic persistent hepatitis, cholestasis, myocardial infarction, injury, hepatic cirrhosis  -fatty liver, primary liver tumour, drug-induced liver demage, metastases to liver, cholangitis, neumathemia, moderate exercise de Ritis index – AST/ALT Ratio AST/ALT: 1. 2. 3. 4. 5. Acute virus hepatitis: < 0,7 uncomplicated, > 0,7 necrotic Hepatic cirrhosis, chronic hepatitis:  1 Myocardoal infarction, injury: >1 Alcoholic hepatitis 2 Chronic agressive hepatitis 4-10 GGT (GGTP)     Gamma-glutamyl transferase is binding with endoplasmic reticulum. Primary task is gamma-glutamyl residue transfer on amino acids or small peptides. Main sources of GGT are epithelial cell of bile ducts, kidney, liver, pancrease, intensine. GGT activity changes are correlated with ALP activity changes. GGT (GGTP)    Increase activity: acute and chronic liver diseases, bile ducts, pancrease, small increase in cardiac infarct. Is induced by barbiturate, phenytoin and estrogen, strong stimulus to in increase activity in urine is alcohol consumption, Activity measurement of GGT is a control factor in alcoholic therapy. Abstinence normalize GGT activity during 2-5 weeks. ALP (AP)– Phosphatase,alkaline Occures in cytoplasmic membranes.  Transmit by membranes phosphatic ion.  ALP – isoforms: * liver ALP * intensine ALP * bile & macromolecular ALP * placental ALP * embryonal cells isoenzyme * renal ALP * bone ALP (b-ALP) Activity ALP increase in wide pathology states. Depends of disease appropriate isoform occures.  ALP (AP) – Reference range Birth Children <1 month 36 – 107 U/L 71 - 213 U/L Children <3 y Children <10 y Adult 71 - 142 U/L 107 - 213 U/L 32 - 92 U/L NH3      Blood ammonia concentration is higher in infant than adults because the development of the hepatic circulation is not completed until after birth. High ammonia concentration ( >60 mol/L)is frequently diagnosed as Reye’s syndrom. Adult patients exhibit elevated blood ammonia in the terminal stages of liver cirrhosis, hepatic failure and acute and subacute liver necrosis. Ammonia is a toxin that acts as a cantral nervous system depressant and can cause coma.  urinary ammonia excretion: acidosis, acute hepatitis, alcoholic hepatitis, urea cycle defect.  urinary ammonia excretion: alkalosis, renal failure, glomerulonephritis, hypercorticoidism, Addison’s disease Bone Profile Total protein  Albumin  Ca P  ALP  Total protein    Plasma proteins serve a number of biological functions in the body. Each protein performs specific functions within this broad categories. Although the exact biological functions of same plasma proteins have not be elucidated, several proteins have become useful biochemical markers of disease. Most plasma proteins, with the exeption of some immunoglobulins and protein hormones, are synthesized in the liver. In a healthy, nondiseased individual, a balance exist between protein anabolism and catabolism(anabolic and catabolicrates are equilibrium and the nitrogen balance is zero). Reference range for total protein: 6.0-8.0 g/dl Protein Trivia The most abundant organic molecule in cells (50% by weight)  30-50K structural genes code for proteins  Each cell contains 3-5K distinct proteins  About 300 proteins have been identified in plasma  Functional diversity of proteins   Structural – Keratin, collagen, actin, myosin Transport – Hemoglobin, transferrin, ceruloplasmin    Hormonal – Insulin, TSH, ACTH, PTH, GH Regulatory – Enzymes What else? Biological functions of protein binding of metals, vitamins, hormones or drugs to proteins serves to trnsport the bound substance to a location where it can be utilized (thyroxine with thyroxine-binding globulin). Maintain The the bound substance in the soluble state (lipid wirh apolipoprotwins). a site for removal of excess of a substance (hemoglobin with hemoplexin). Provide Prevent loss of substances through the kidneys (iron with transferrin). Acute phase react. response. Immune The composition of proteins   Amino acids (simple proteins) – 20 common (standard) amino acids Conjugated proteins contain a prosthetic group: – – – – – Metalloproteins Glycoproteins Phosphoproteins Lipoproteins Nucleoproteins The size of proteins An arbitrary lower limit is a MW of 5,000  Proteins can have MW greater than 1 million, although most proteins fall in the range of 12-36K  – 100-300 amino acids – Albumin (the most abundant protein in humans) is 66K and contains 550 amino acids (residues) Protein structure     Primary structure – Amino acid sequence Secondary structure – -helix or random coil Tertiary structure – 3-D conformation (globular, fibrous) Quaternary structure – Multi-protein assemblies Amino acids (1º structure) The amino acid sequence is the only genetically-stored information about a protein  Each amino acid is specified by a combination of 3 nucleic acids (codon) in mRNA:  – e.g., CGU=Arg; GGA=Gly; UUU=Phe Properties of amino acids O H2N CH R C OH + O H3N CH R C O- Undissociated form  Zwitterion (dipolar) The –R group determines, for the most part, the properties of the amino acid  Substances that can either donate or accept a proton are called ampholytes Acidic and basic amino acids  Acidic – Asp R=CH2COO– Glu R=(CH2)2COO-  Basic – Lys R=(CH2)4NH3+ – Arg R= (CH2)3NHC(NH2)2+ – His R: NH CH2 N + 2 Uncharged amino acids   Non-polar (hydrophobic) amino acids – Ala, Val, Leu, Ile, Pro, Phe, Trp, Met Polar (hydrophilic) amino acids – Gly, Ser, Thr, Cys, Tyr, Asn, Gln Stereochemistry of amino acids R R NH2 H COOH HOOC H NH2 L-Alanine D-Alanine  All naturally-occurring amino acids found in proteins have the “L” configuration Essential amino acids  Humans ordinarily cannot synthesize: – Leu, Ile, Val, Met, Phe, Trp, Thr, Lys, His (Arg)  Dietary protein is the principal source of essential amino acids The peptide bond O H2N CH R C OH H2 O H2N CH R O C OH The peptide bond O H2N CH R C N H CH R O C OH Dipeptide Amino acid composition and protein properties The –R groups determine, for the most part, the properties of the protein  Proteins rich in Asp, Glu are acidic (albumin is an example)  Post-translational modifications of proteins have significant effects on their properties, as well.  Coiling (2 structure)    Linus Pauling described the -helical structure of proteins Pro and OH-Pro break the -helix Ser, Ile, Thr, Glu, Asp, Lys, Arg, and Gly destabilize the -helix Folding (3 structure)   J. C. Kendrew deduced the structure of myoglobin from Xray crystallographic data Globular proteins have stable 3-dimensional conformations at physiological pH, temperature Myoglobin   Protein 3 structure is influenced by  and  regions Proteins fold in order to expose hydrophilic regions, and sequester hydrophobic regions 4 structure  Hemoglobin has 4 subunits – Two  chains – Two  chains  Many enzymes have quaternary structures Measuring proteins   By reactivity – Biuret reaction, Lowry method By chemical properties – Absorption at =260 nm (Phe) or 280 nm (Tyr, Trp)   By activity – Enzymes, immunoglobulins By immunogenicity Separating plasma proteins  Chromatography – Gel (size exclusion), HPLC, ion exchange, immunoaffinity  Electrophoresis – Starch gel, agarose gel, cellulose acetate, PAGE Serum protein electrophoresis Albumin 1 2   + - Albumin  Most abundant protein in plasma (approximately half of total protein) – Synthesized in liver – t½=15-19 days  Principal functions – – – – Maintaining fluid balance Carrier Anti-oxidant activity Buffer Albumin Serves as transport protein for fatty acids, bilirubin, etc., and maintains oncotic pressure. the fastest on celulose acetate electrophoresis. Migrates Generally used on a marker of nutritional status. in urine to follow proteinuria. Measured Found in highest concentration in plasma. Increased values occur dehydration. Albumin Decreased values occur in malnutrition, starvation, malabsorption and protein losing disorders (nephrotic syndrome), liver disease.  Loss in urine – renal disease, especially glomerular kidney damage.  Retinol binding protein. Methods for quantify serum, plasma or urine albumin currently include electrophoresis, immunochemical and dye-binding procedure. Reference range: 29 – 55 g/l – (0-1 y) 35 – 50 g/l – (1-31y) Values begin to decline after age 40 Clinical significance of albumin Hyperalbuminemia is rare and of no clinical significance  Hypoalbuminemia  – Increased loss (nephrotic syndrome) – Decreased production (nutritional deficit, liver failure) Analbuminemia  Bisalbuminemia, dimeric albumin  Pre-albumin  Thyroxine-binding protein (not an incipient form of albumin), also called transthyretin, or TBPA – Also complexes with retinol-binding protein (RBP) Only protein that migrates anodal to albumin  Sensitive marker of nutritional status, since its t½ is only 2 days  1-Antitrypsin Protease inhibitor that binds to, and inactivates, trypsin  Deficiency is associated with  – Pulmonary emphysema – Cirrhosis  SPE is only a screening test for AAT deficiency Other 1 proteins   1-Acid glycoprotein (orosomucoid) – Biological function is unknown 1-Fetoprotein (AFP) – Principal fetal protein, used to screen for fetal abnormalities (neural tube defects) 2-Macroglobulin Largest non-immunoglobulin in plasma  Protease inhibitor  Increased in nephrotic syndrome (size)  Complete genetic deficiency is unknown  (2) Ceruloplasmin Copper transport protein  Participates in plasma redox reactions  Cp levels fluctuate with a variety of physiological states, but measurement is usually to screen for Wilson’s disease  – Plasma Cp is decreased due to inhibition of synthesis (2) Haptoglobin  Binds to, and preserves, hemoglobin but not myoglobin – Complex also has peroxidase activity, and may be involved in inflammatory response  Hemolytic diseases can deplete Hp levels () Transferrin Iron transport protein, and also binds copper  Transferrin is increased in iron deficiency anemia, as well as pregnancy and estrogen therapy  Decreased in inflammation, malignancy, or liver disease  2-Microglobulin Small protein (MW=11.8K)  BMG is filtered in the glomerulus, but is reabsorbed in the renal tubules.  – Urinary BMG levels are a sensitive measure of renal tubular function  Increased in renal failure () Compliment proteins C3 and C4 migrate in the  region  Compliment proteins are decreased in genetic deficiencies, and increased in inflammation.   Region Includes immunoglobulins (IgG, IgA, IgM) and C-reactive protein  Single sharp peak is indicates a paraprotein associated with a monoclonal gammopathy (multiple myeloma)  CRP is the most sensitive indicator of Acute Phase Reaction  – Inflammation, trauma, infection, etc. X upper limit of normal Acute Phase Reactants 10 C-reactive protein 5 1-Antitripsin 1 C3 5 1 2 3 Days 4  Other ACPs include 1-acid glycoprotein, haptoglobin, and ceruloplasmin Normal SPE Albumin 1 2   Immediate response pattern Decrease in albumin Increase in APR haptoglobin Albumin 1 2   Delayed response pattern Albumin decreased Haptoglobin increased Gamma globulins increased Albumin 1 2   Hypogammaglobulinemia Decreased gamma globulins Albumin 1 2   Nephrotic Syndrome Decreased albumin Increased 2-macroglobulin Decreased gamma globulins Albumin 1 2   Hepatic cirrhosis Decreased albumin (synthesis) Increased gamma globulins (polyclonal gammopathy) “- bridging” Albumin 1 2   Monoclonal gammopathy Albumin decreased Sharp peak in gamma region Albumin 1 2   Protein-losing enteropathy Decreased albumin Decreased gamma globulins Increased 2-macroglobulin Albumin 1 2   Biological functions of protein       The binding of metals, vitamins, hormones or drugs to proteins serves to trnsport the bound substance to a location where it can be utilized (thyroxine with thyroxine-binding globulin). Maintain the bound substance in the soluble state (lipid wirh apolipoprotwins). Provide a site for removal of excess of a substance (hemoglobin with hemoplexin). Prevent loss of substances through the kidneys (iron with transferrin). Acute phase react. Immune response. Selected methodologies for serum total protein quantitation Kjeldahl method  Biuret method  Lowry method  Refractometry method  Ca - Calcium      Approximately 40% of calcium is protein bound, mostly to albumin. The remainder of calcium is free (46%) and complexed as organic and inorganic salts. Concentration of Ca vary with age and are highest in the neonatal period. Upright position for 15 min causes an increase of 4-7% in Ca level. Total serum Ca level is alerted by changes in protein concentration. False elevations of serum Ca are caused by venous stasis during collection and by prolonged storage of blood. Ca – Calcium Total (serum) Newborn 0-10 days Newborn 10 d – 1 y Children 1–12 y Children 12-18 y Adults 18-60 y Adults 60-90 y 7,6 – 10,4 mg/dL 9,0 – 11,0 mg/dL 8,8 – 10,8 mg/dL 8,4 – 10,2 mg/dL 8,6 – 10,0 mg/dL 8,8 - 10,2 mg/dL Adults >90 y 8,2 – 9,6 mg/dL Calcium Ionized (serum) Newborn < 5 days Youth 4,4 – 5,92 mg/dL 4,8 – 5,52 mg/dL Adult 4,64 – 5,28 mg/dL Ca – Calcium Total    - sarcoidosis, malignant disease with bone involvement, malignant disease without bone involvement, milk-alkali syndrom, primary and tetrinary hyperparathyroidism, osteitis fibrosa.  - osteomalacia, vitamin D deficiency, pseudo and secondary hypoparathyroidism, magnesium deficiency, chronic renal failure, massive blood transfusion, alcoholism, neonatal prematurity Methods of calcium analysis 1. Total calcium: a. Atomic absorption spectrophotometry b. Spectrophotometric c. Fluorometric complexpmetry 2. Ionized calcium: Ion-Selective Electrode (ISE) P.- Phosphorus  Ubiquitous cellular substance. The plasma concentration represent the equilibrum between dietary intake, compartmental distribution within the body, renal tubular reasorption and excretion by kidneys. Disturbance of this equilibrium may produce either hypo- or hyperphosphatemia. Methods of phosphorus analysis: phosphomolybdate reduction, enzymatic.  P.- Phosphorus Newborns 0-10 d 10 d – 2 y Children 2 y – 12 y Children and adults 12-60 y Adults >60 y male Adults >60 y female 4,5 – 9,0 mg/dL 4,5 – 6,7 mg/dL 4,5 – 5,5 mg/dL 2,7 – 4,5 mg/dL 2,3 – 3,7 mg/dL 2,8 – 4,1 mg/dL P.- Phosphorus    - renal insufficiency,osteolytic metastatic bone tumors, sarcoidosis, pseudohyporparathyroidism, milk-alkali syndrome, vitamin D intoxication, acromegaly, lactic acidosis, respiratory acidosis.  - primary hyperparathyroidism, osteomalacia, renal tubular acidisis, growth hormone deficiency, acute alcoholism, hypokalemia, severe diarrhea, vomiting, acute gout, severe malnutrition, respiratory alkalosis. The Heart Aorta Superior vena cava Pulmonary arteries RA LA LV RV The Heart (posterior view) Aorta Pulmonary arteries Superior vena cava Pulmonary veins Inferior vena cava Normal Electrocardiogram R T U P Q S Myocardial infarction Left coronary artery Anterior left ventricle Right coronary artery ECG changes in myocardial infarction R S-T elevation T P Q S Heart profile ALT & AST  CK  Myoglobin  Troponin T and I  LDH K  History of cardiac markers       1975: Galen describes the use of CK, LD, and isoenzymes in the diagnosis of myocardial infarction. 1980: Automated methods for CK-MB (activity) and LD-1 become available. 1985: CK-MB isoforms are introduced. 1989: Heterogeneous immunoassays for CKMB (mass) become available. 1991: Troponin T immunoassay is introduced. 1992: Troponin I immunoassay is introduced. CK – Creatine Kinase         CK is primary foumd in muscle tissue. Significant increase of CK occur in serum when either skeletal muscle or cardiac muscle has been damage. CK isoenzymes: CK-MM, CK-MB, CK-BB.  CK-MM: auto accident, dystrophy, exercise  CK-MM and CK-MB: myocardial infarction, heart anoxia, surgery. CK-BB is found in serum from patints with tumors, particulary those of the gonads; infarction of intestine or kidney. In a healthy, nondiseased individual CK-BB is undetectable CK: steroid therapy, hyperthyroidism. Analysis method: electrophoresis, immunoinhibition, immunoassay. The average time for maximum elevation for CK in patients with a myocardial infraction has been reported as: CK-MB 6-12h, total CK:18-24h. Myoglobin      O2-binding cytosolic protein found in all muscle tissue (functional and structural analog of hemoglobin) Low molecular weight (17,800 daltons) Elevations detected within 1-4 hours after symptoms; returns to normal after 12 hours Nonspecific but sensitive marker--primarily used for negative predictive value Usually measured by sandwich, nephelometric, turbidimetric, or fluorescence immunoassay Troponin I   High concentration of skeletal muscle (>750 g/L). Cardiac troponin I is one of three proteins found in the troponin regulatory complex. The troponin complex mediates the interaction of actin and myosin through alternations of tropomyosin positions in the groove between the two actiwe strands. Cardiac troponin I has a molecular weight of about 23 000 and it existi in three distinct isoforms found in fast and slow-twitch skeletal muscle and in cardiac muscle. Human cardiac troponin I has 60 % sequence homology with the other isoforms and furthermore, has 31 additional amino acid residues at its N-terminus. Therefore, its unique structure and known early release from damaged myocardium make it a promising cardiac marker. Indeed, troponin I is potentially a more specific marker for cardiac damage than troponin T. Troponin I Methods of analysis: RIA, ELISA.  Reference range: RIA <10 g/L ELISA 3,1 g/L  - Myocardial infarction  - Chronic ischemic heart disease, acute skeletal muscle injury  Troponin T  Exists in two corpantments. About 6% is in the soluble cytosolic pool, and the remainder is bound to the thin filament in the sarcomere. This compartmentalization may explain the biphasic release ptofile of troponin T folowing cardiac damage LDH – lactate dehydrogenase   Is found in all tissues and has a distribution pattern in plasma or serum in which LD2 is predominant isoenzyme. In this pattern, LD1 is about 50-75% of LD2 and lesser amounts in order are LD3, LD4 and LD5. All five of the isoenzymes are present in clinical samples, although the absolute amounts of each isoenzyme varies. Heart muscle has significant amount of LD1 and LD2 with about twice as much LD1 as LD2. This is the only tissue with LD1 > LD2. Liver, skeletal muscle and skin contain predominatly LD5 with some LD4 and traces of other three isoenzymes. Lung contains a majority of LD3 but it has large amounts of all the other isoenzyme fractions as well. LDH – lactate dehydrogenase   The combination LDH isoenzyme and CK analyses is more pawerful than either determination alone, since these result-whichare samewhat independent measures of cardiac damage – tend to support one another. The average time for maximum elevation for a series of enzymes and isoenzymes in patients with a myocardial infraction has been reported as: CK-MB 6-12h, total CK:18-24h and LDH 48-72h. LDH1 appears to be less than LDH2 in early samples when CK-MB rising. But as the total LDH concentration increases, both LDH1 and LDH2 increase in serum.Eventually, LDH1 / LDH2 ratio flips so the LDH1 concentration is greater than the LDH2. As the time after the clinical event increases the LDH1 concentration eventually returns after several days to values lover than LDH2. LDH – methods of analysis Electrophoresis  Immunoinhibition  Immunoassay  Lipid profile Total cholesterol  HDL-cholesterol  triglyceride  Adapted from AstraZeneca education material Classification of Lipoproteins Based on density: 1. Chylomicrons 2. Very low-density lipoprotein (VLDL) 3. Intermediate-density lipoprotein (IDL) 4. Low-density lipoprotein (LDL) 5. High-density lipoprotein (HDL) LDL-Cholesterol Risk associated with LDL-C is increased by other risk factors: – – – – low HDL-cholesterol smoking hypertension diabetes Triglycerides 1. 2. 3. 4. 5. Associated with increased risk of CHD events Link with increased CHD risk is complex – may be related to:  low HDL levels  highly atherogenic forms of LDL-cholesterol  hyperinsulinaemia/insulin resistance  procoagulation state  hypertension  abdominal obesity May have accompanying dyslipidaemias Normal triglyceride levels <150 mg/dL Very high triglycerides (>1000 mg/dL,11.3 mmol/L) increase pancreatitis risk HDL-Cholesterol     HDL-cholesterol has a protective effect for risk of atherosclerosis and CHD The lower the HDL-cholesterol level, the higher the risk for atherosclerosis and CHD – low level (<40 mg/dL) increases risk HDL-cholesterol tends to be low when triglycerides are high HDL-cholesterol is lowered by smoking, obesity and physical inactivity Adapted from AstraZeneca education materia Adapted from AstraZeneca education materia Adapted from AstraZeneca education materia Thyroid profile  TSH – thyroid stymulating hormone T4 – tyroxine (L- tetraidothyronine)  TSH - thyroid stymulating hormone The main functions of the thyroid gland are the synthesis, storage and secretion of thyroid hormones. Each function is regulated through the action of TSH produced by the anterior pituitary gland TSH - thyroid stymulating hormone   TSH measurements with sufficient sensitivity to distinguish low levels from normal have become the preferred first line test for hyper- and hypothyroidism. The high sensitivity test appeares to be especially useful in distinguishing sick euthyroid patients: in differentiating mild, subclinical hyperthyroidism from over Graves’ disease; in monitoring thyroid cancer patients on thyroxine; and in monitoring the adequacy of thyroid hormone replacment in hypothyroid patients. If basal serum TSH levels are detectable by sensitive assay, then no diagnostic benefits is gained by performing a TRH (thyrotropin-releasing hormone)stimulation test. Test for measuring TSH – RIA, IRMA TSH – reference range U/mL 1 - 39 1,7 – 9,1 0,7 – 6,4 0,4 – 4,2 Newborn: 1- 4 d Newborn: 2-20 wk Children: 20 mo – 20 y Adults: 21 – 54 y Adults: 55 – 87 y Pregnancy: first trimestr Pregnancy: second trimestr Pregnancy: third trimestr 0,5 – 8,9 0,3 – 4,5 0,5 – 4,6 0,8 – 5,2 TSH – diagnostic information  primary hypothyroidism (3 to 100 times normal), Hasimoto’s thyroidism with clinical hypothyroidism and 33 % of patient with Hasimoto’s thyroidism who are clinically euthyroid, ectopic TSH secretion (lung, brest tumors), subacute thyroiditis (recovery phase), nonthyroidal illness (recovery phase), thyroid hormone resistance (pituitary), tertiary hypothyroidism (hypothalamic), subclinical hyperthyroidism (e.g.,toxic multinodular goiter, autonomous thyroid hormone secretion, exogenous thyroid hormone therapy, treated Graves’ disease, ophthalmophaty of euthyroi Graves’ disease), euthyroid sick syndrome  - primary hyperthyroidism, secondary hypothyroidism T4, thyroxine (L- tetraidothyronine)    Is produced by thyroid gland, is indispesable for normal mental and physical growth and development. Is involved in regulation of many cellular and metabolic process. T4 accounts for approximately 97% of circulating thyroid hormones. Test for measuring T4 – RIA, FAPIA, luminoscence assay T4, thyroxine – reference range g/dL Newborn: 1 –3 d 11,8 – 22,6 5,6 – 15 4,6 – 10,5 male 5,5 – 11 female Children: 1wk – 12 mo 7,2 – 16,6 Children: 1-15 y Adult T4, thyroxine – diagnosis information   - hyperthyroidism; state with increased TBG (thyroxine-binding globulin) – pregnancy,genetically increased TBG, acute intermittent porphyria, primary biliary cirrhosis; thyrotoxicosis factitia, same cases of acute thyroiditis, hepatitis (by 4 wk), obesity, acute psychiatric illnesses, hyperemesis gravidarum   - hypothyroidism; state with decreased TBG (nephrotic syndrom, chronic liver disease, genetically decrease TBG, malnutrition), panhypopituitarism, strenuouse exercise. Pancreatic profile    Amylase Lipase Acute pancreatitis Pancretitis occurs in acute nad chronic forms.Acute pancretitis is a condition in which a sudden, noninfectiouse inflammations develps in the pancreas, cousing extreme abdominal pain. The condition is often spontaneos but may be brought on by alcoholic excess and hyperlipidemia. The time-honored laboratory tests for diagnosis of pancreatitis are measurement of serum and urine amylase and serum lipase. Amylase  The serum amylase rises rapidly after the onset of the attack in 1 to 2 hours. The extent of the elecation is roughly proportional to the severity of pancreatitis. 90% of persons with significant pancreatitis will exhibit increased amylase. Milder cases of acute pancreatitis may have equivocal increases in the serum amylase level. It is often useful in these situations to collect timed urine samples and measure the amylase content of the urine. Amylase Serum Urine U/L 10 - 53 < 290  - Acute pancreatitis,  - Chronic pancreatitis, carcinoma of pancreatic,  - Chronic renal failure, parotitis, ectopic pregnancy, perforated stomach, hepatitis and variety of intraabdominal catastrophes. Lipase   The serum concentration of lipase also rises during pancreatitis. The serum lipase rises with much the same pattern as amylase, thout the increase is not as great. There is good evidence that lipase is increased in some of the pancreatitis patients who do not show elevated amylase value. The lipase is more specific than amylase. Pancreatitis is only entity that will cause an increase in lipase.In contrast, amylase levels will increase in renal failure, hepatitis etc. Lipase Children < 16 y U/L < 78 Adult  - Acute and chronic pancreatitis,  - Carcinoma of pancreatic. < 190 Diabetes Mellitus    A syndrome characterized by hyperglycemia resulting from absolute or relative impairment in insulin secretion and/or insulin action. Diabetes attributed to pancreatic disease Diabetes associated with other endocrine diseases: Type II DM can be secondary to Cushing's syndrome, acromegaly, pheochromocytoma, glucagonoma, primary aldosteronism, or somatostatinoma. . The prevalence of type I DM is increased in patients with certain autoimmune endocrine diseases, eg, Graves' disease, Hashimoto's thyroiditis, and idiopathic Addison's disease. Diabetes Mellitus Comparison of two type of diabetes mellitus IDDM Defect or deficiency -cells destroyed, eliminating production of insulin. Common Low to absent Ketoacidosis Unresponsive Always necessary NIDDM Inability of -cells to produce appropriate quantities of insulin; insulin resistance. Rare Normal to high Hyperosmolar coma Responsive Usually not required Ketosis Plasma insulin Acute complications Oral hypoglycemic drugs Treatment with insulin Diabetes Mellitus    Late complications: retinopathy, nephropathy, polyneuropathy, mononeuropathies, autonomic neuropathy, the risk of infection from fungi and bacteria is increased. Diagnosis: plasma glucose levels of > 126 mg/dL ( > 6.99 mmol/L) be considered diagnostic for DM. An oral glucose tolerance test (OGTT) may be helpful in diagnosing type II DM in patients whose fasting glucose is between 115 and 140 mg/dL (6.38 and 7.77 mmol/L) and in those with a clinical condition that might be related to undiagnosed DM (eg, polyneuropathy, retinopathy). However, various conditions other than DM, such as effects of drugs, and normal aging can cause abnormalities in the OGTT. Diabetes Mellitus   Diet to achieve weight reduction is most important in overweight patients with type II DM. If improvement in hyperglycemia is not achieved by diet, trial with an oral drug should be started. Plasma glucose monitoring: All patients should learn self-monitoring of glucose, and insulin-treated patients should be taught to adjust their insulin doses accordingly. Glucose levels can be tested with easy-to-use home analyzers using a drop of fingertip blood. Glycosylated hemoglobin Hb A1c  Most physicians periodically determine glycosylated hemoglobin (Hb A1c) to estimate plasma glucose control during the preceding 1 to 3 mo. Hb A1c is the stable product of nonenzymatic glycosylation of the -chain of Hb by plasma glucose and is formed at rates that increase with increasing plasma glucose levels. In most laboratories, the normal Hb A1c level is about 6%; in poorly controlled diabetics, the level ranges from 9 to 12%. Hb A1c is not a specific test for diagnosing diabetes; however, elevated Hb A1c often indicates existing diabetes. Major metabolic events that leads to hyperglycemia and ketoacidosis in DM Insulin,Glucagon  Breakdown of tissue proteins  Gluconeogenesis  Glucogenolysis  Lipolysis  Free fatty acids  Hepatic output of glucose  Glucose uptake by tissues in plasma  Hepatic output of ketone bodies Hyperglycemia Ketoacidosis Control profile Na, K, Cl,  urea, creatinine, bilirubin,  AST, ALT, ALP, GGTP,  Albumin, total protein  Ca, P, uric acid  Acute-Phase Protein Any protein whose plasma concentration increases (or decreases) by 25% or more during certain inflammatory disorders. The acute-phase proteins include:  CRP – C-reactive protein  SAA or SAP– serum amyloid A or P  Fibrynogen  Alpha 1-acid glucoprotein,  Haptoglobin,  Ceruloplasmin. CRP        is produced in the liver; those cells produce CRP detectable on lymphocytes. recognize specifically foreign pathogens and damaged cells of the host and to initiate their elimination by interacting with humoral and cellular effector systems in the blood. CRP binds with high affinity to chromatin. It has been proposed that one of its major physiologic functions is to act as a scavenger for chromatin released by dead cells during the acute inflammatory process. CRP molecule has both a recognition and an effector function. C-reactive protein are predictive of cardiovascular events in patients with coronary heart disease. C-reactive protein not only may be a marker of low-grade chronic systemic inflammation but also may be directly involved in atherosclerosis. It can amplify the antiinflammatory response through complement activation, tissue damage, and activation of endothelial cells. Reference range: <0,5 mg/dL SAP SAP neutralizes LPS and it is potentially useful in defense against serious gram-negative sepsis.