ADDITIONAL REVIEW NOTES CLINCAL CHEMISTRY 1. CONVERSION OF TRADITIONAL UNITS TO SI UNITS FOR COMMON CHEMISTRY ANALYTES. CONVENTIONAL | —SI__| CONVERSION CONVENTIONAL | SI__| CONVERSION CURRENT | _UNITS | FACTOR’ CURRENT | UNITS | FACTOR ‘Albumin | g/00 mL gi | 10 Tron mgfdL mov | 0.178 ‘AST UML (mu/mly kati | 0.0167 Lithium mEg/L mov [1.0 ‘Ammonia | wa/dl mol _| 0.587 Magnesium | mEq/L mmol_| 0.8 Bicarbonate | mEq/L mmoll_| 1.0 Osmolality | mOsm/kg mmolkg | 1.0 Bilirubin | mg/d. umol_[ 47.4 Phosphorus _| mg/dL. mmolL_| 0.323 BUN mg/d mmol/L | 0.357 Potassium _| mEq/L ‘mmol/L 4.0 Calcium | mg/dl. mmol_| 0.25 ‘Sodium | mEq/L mmol/L 4.0 Chloride | mEq/L. ‘mmoW/L_| 1.0 Thyroxine | g/d nmovL | 12.8 Cholesterol | mg/dL mmol/L | 0.026, Total ‘gidL. ofl 10 | protein | | Cortisol | gil pmol | 0.0276 Triglyceride | mg/dL [mmol | 0.0113 Creatinine | mg/dL moll | 88.4 Uric acid | mg/d [mmolit_| 0.0595 Crea mUmin mus | 0.0167 Vit B12 nigimt. pmov | 0.0738 clearance Folic acid | ngimt mov | 2.27 PCO: mmiFig kPa__| 0.433 Glucose | mg/dL ‘mmol/L | 0.0555 PO: mmiFig kPa__| 0.133 Hemoglobin | g/dL. gh [10 2. Visible light falls in between, with the color violet at 400-nm and red at 700-nm wavelengths being the approximate limits ofthe visible spectrum. (Bishop) 3,_ EXAMPLES OF NONIONIZING RADIATION IN CLINICAL LABORATORIES TYPE ‘APPROXIMATE ‘SOURCE EQUIPMENT | PROTECTIVE MEASURES WAVELENGTH EXAMPLE Tow frequency Tom’ Radiofrequency coil in GP- _ | Engineered shielding and mass spectrometer posted pacemaker warning Microwaves 3m-3mm Energy-beam microwave | Engineered shielding used to accelerate tissue staining in histology-prep processes Infrared 750 nm-0.3 em Heat lamp, laser Containment and appropriate warning labels Visible spectrum “400 - 750 nm General illumination and Filters, diffusers, and glare nonreflective surfaces Ultraviolet “4 400 nm Germicidal lamps used in| Eye and skin protection; UV biologic safety cabinet warning labels 4, BIOCHEMICAL FUNCTIONS OF THE KIDNEY FUNCTION. EXAMPLE ‘Synthesis Erythropoietin, rennin, prostaglandins Metabolism Inactivation of aldosterone, glucagons, insulin; activation of vitamin D, formation of creatine Excretion "Ammonia, urea, uric acid; several minerals; toxic substances 5. __ APPROACHES TO ASSAY OF UREA NITROGEN (Calbreath) METHODS ‘COMMENTS Colorimetric: diacetyl Inexpensive, lacks specificity Enzymatic: NHs formation Greater specificity, more expensive * Isotope dilution mass spectrometry - reference method 6 renal failure is called uremia, or the uremic syndrome. ‘An elevated concentration of urea in the blood is called azotemia. Very high plasma urea concentration accompanied by This condition is eventually fatal if not treated by dialysis or transplantation. Conditions causing increased plasma urea are classified according to cause into three main categories: Prerenal, renal, and postrenal. (Bishop) 7.__APPROACHES TO ASSAY OF CREATININE (Calbreath) METHODS COMMENTS Colorimetric: end point ‘Simple, nonspecific Colorimetric: Kinetic Rapid, increased specificity Enzymatic Measure ammonia colorimetrically or with electrode ion-selective 8 The methods most frequently used to measure creatinine are based on the Jaffe reaction. In this reaction, creatinine reacts with picric acid in alkaline solution to form a red-orange chromogen, The reaction was adopted for the measurement of blood creatinine by Folin and Wu. The reaction is nonspecific and subject to positive interference by a large number of compounds, including acetoacetate, acetone, ascorbate, glucose, and pyruvate. More accurate results are obtained when creatinine in a protein-free filtrate is adsorbed onto Fuller's earth (aluminum magnesium silicate) or Lloyd's reagent (sodium aluminum silicate) then eluted and reacted with alkaline picrate.(Bishop)Page |2 9. APPROACHES TO ASSAY OF URIC ACID (Calbreath) METHODS COMMENTS Colorimetric Problems with turbidily, several common drugs interfere Enzymatic: UV Need special instrumentation and optical cells Enzymatic: H02 Interference by reducing substances 10. LIVER FUNCTIONS FUNCTION EXAMPLES ‘Synthesis Proteins — albumin, cholinesterase, coagulation proteins, cholesterol, bile salts and glycogen Metabolism Glucose to acetyCoA, gluconeogenesis, amino acid conversions, fatty acids Detoxification Bilirubin, drugs, ammonia Excretion Bile acids 11. Cigarette smoking by the patient is a significant source of ammonia contamination. It is recommended that patients do not ‘smoke for several hours before the sample is collected. (Bishop) 12. BILIRUBIN FRACTIONS Conjugated bilirubin | Contains one or two attached glucuronic acid molecules Reacts directly with the color reagent Also referred to as DIRECT BILIRUBIN Unconjugated bilirubin | Noncovalently attached to albumin Does not react with the color reagent unti the bilirubin is first dissociated from the protein INDIRECT BILIRUBIN Delta bilirubin Bilrubin fraction that is covalently attached to protein Reacts directly with the color reagent and contributes to the direct, or conjugated, bilirubin value 13. COLOR REACTION FOR QUANTITATION OF BILIRUBIN Bilirubin + diazotized sulfanilic acid -> azobilirubin + _ Color is proportional to the concentration of bilirubin ASSAY T EVELYN-MALLOY T JENDRASSIK-GROF pH ‘Acid ‘Alkaline Dissociating agent Methanol Caffeine-sodium benzoate Diazo product Red or reddish-purple color Blue (maximum absorbance around 600 {absorption maximum in the region | nm) of 560 nm) 14. Gilbert disease, Crigler-Naljar syndrome, and physiologic jaundice of the newborn are hepatic causes of jaundice that result in elevations in unconjugated bilirubin, Conditions such as Dubin-Johnson and Rotor syndrome are hepatic causes of jaundice that result in elevations in conjugated bilirubin. (Bishop) 15. In the more serious or type I form of the Crigler-Naljar syndrome, the unconjugated hyperbilirubinemia becomes marked, almost always exceeding 5 mg/dL. and causing jaundice, and sometimes exceeding 20 mg/dL. Affected infants develop severe unconjugated hyperbilirubinemia, which typically leads to kernicterus, deposition of bilirubin in the brain, particularly affecting the basal ganglia, mainly the lenticular nucleus, causing severe motor dysfunction and retardation, The danger of kernicterus is a certainty at levels exceeding 20 mg/dL. It is vital to treat these infants with phototherapy to cause excretion of the unconjugated bilirubin. (Henry) 16. When serum bilirubin approaches 430 mmol/L. (25 mg/L), interference may be observed in assays for albumin (4- hydroxyazobenzene-2-carboxylic acid [HABA] procedure), cholesterol (using ferric chloride reagents), and total protein (Biuret procedure). (Henry) 17. CHARACTERISTICS OF SELECTED PLASMA PROTEINS (Bishop 6" Ed.) Prealbumin (Transthyretin) | INDICATOR OF MALNUTRITION; binds thyroid hormones and relinol-binding protein ‘Albumin Binds bilirubin, steroids. fatty acids; major contributor to oncotic pressure ‘ai-Globulins ‘at-antitrypsin ‘Acute-phase reactant; protease inhibitor ‘at-fetoprotein Principal fetal_protein ‘at-acid glycoprotein ‘Acute-phase reactant | (orosomucoid) |a1-lipoprotein (HDL) ‘Transports lipid [ai-antichymotrypsi Inhibits serine proteinases (i, chymotrypsin) [nter-a-trypsin Inhibits proteinases (le, trypsin) Ge-globulin Binds vitamin D and actinPage |3 @2-Globulins Haptoglobins ‘Acute-phase reactant; binds hemogiobin Ceruloplasmin Peroxidase activity: contains copper ‘«2-Macroglobulin Inhibits thrombin, trypsin, pepsin f-Globulins Pre-f-Lipoproteins (VLDL) _| Transpors lipids (primarily triglyceride) ‘Transferrin (Siderophilin) | Transports iron Hemopexin Binds heme {-Lipoproteins (LDL) ‘Transports lipids (primarily cholesterol) {f2-Micorglobulin (B2M) ‘Component of human leukocyte antigen (HLA) molecules class | ‘Complement Immune response Fibrinogen Precursor of fibrin clot C-reactive protein (CRP) | Acute-phase reactant; motivates phagocytosis in inflammatory disease (Bishop) (Henry-y) 7-Globulins Immunoglobulin G Immunoglobulin A Immunoglobulin M ‘Antibodies (early response) Immunoglobulin D ‘Antibodies Immunoglobulin & Antibodies (allergy) 18. SIGNIFICANT PROTEINS ELECTROPHORETIC FRACTIONS FRACTION ‘SPECIFIC PROTEINS ‘Albumin Albumin ‘Alpha; globulin Alpha antitrypsin, lipoproteins ‘Alpha, globulin Ceruloplasmin, haptoglobin, alpha, macroglobulin, lipoproteins Beta globulin ‘Transferrin, hemopexin, complement system, lipoproteins Gamma globulin immunoglobulins i i t rt tit |Page |4 19. Four major lipoprotein classes have been identified: Chylomicrons (CMs), very-low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL). Several minor lipoproteins have also been identified, including intermediate-density lipoprotein (IDL) and lipoprotein(a) (Lpfal) 20. MAJOR CLASSES OF HUMAN PLASMA LIPOPROTEINS: CHEMICAL COMPOSITION Protein (%) Cholesterol(%) | Cholesteryl | Triglyceride (%) | Phospholipid (%) Esters (%) Chylomicrons 1-2 1-3 2-4 80-98 3-6 VLDL 6-10 4-8 16-22 45-65 715-20 TDL Intermediate between VLDL and LDL LOL 18-22 6-8 45 - 50 4-8 28-24 HDL 45-55 3-5 15-20 2-7 26 - 32 CHARACTERISTICS OF HUMAN APOLIPOPROTEINS AND THEIR VARIANTS (Bishop 3" Ed.) APOLIPOPROTEINS | FUNCTIONS MAJOR SOURCE ‘Apo A-l Major structural protein in HDL Liver and intestine Activates LCAT Ligand for HDL binding ‘Apo Act ‘Structural protein in HDL Liver Activates LCAT Enhances hepatic triglyceride lipase activity ‘Apo AW ‘Component of intestinal ipoproteins Intestine ‘Apo B-100 Major structural protein in VLDL and LDL Liver Ligand for the LDL receptor ‘Apo B48 Primarily structural protein in chylomicrons intestine ‘Apo C-1 Activates lipoprotein lipase Liver ‘Apo C-Il Activates lipoprotein lipase Liver Activates LCAT ‘Apo Cell Inhibits ipoprotein lipase Liver | inhibits receptor recognition of apo E ‘Apo E2,3.4 | Binds to LDL-receptor and remnant-eceptor Liver ‘Apo (a) ‘Structural protein for Lp (a) Liver May inhibit plasminogen binding *LCAT, lecithin-cholesterol acyltransferase “There are many minor apolipoproteins, such as apo D, apo J, apo H, apo F, and apo G. BLOOD LIPOPROTEIN PATTERNS IN PATIENTS WITH HYPERLIPOPROTEINEMIA TYPE LIPOPROTEIN PATTERN" 1 Extremely elevated TG due to the presence of chylomicrons ila Elevated LOL Ub, Elevated LDL and VLDL WL Elevated cholesterol, TG; presence of (-VLDL Vv Elevated VLDL. Vv Elevated VLDL and presence of chylomicrons 21. B-VLDL (‘floating 6" lipoprotein) is an abnormal lipoprotein that accumulates in type 3 hypertipoproteinemia. It is richer in cholesterol than VLDL and apparently results from the defective catabolism of VLDL. The particle is found in the VLDL density range but migrates electrophoretically with or near LDL. (Henry) 22. Lp(a) has a density similar to LDL, but migrates similarly to VLDL on electrophoresis. Thus it can be detected when the d > 1.008 gimL protein is examined electrophoretically. When Lp(a) is present in concentrations exceeding 20-30 mg/dL. (Le., when it contributes more than about 10 mg/dL to the LDL-C measurement) an additional band with pre-B mobility is also observed in the d > 1.006 kg/L fraction (hence the name sinking pre-f-lipoprotein). (Henry) 23. LpX is an abnormal lipoprotein found in patients with obstructive biliary disease, and in patients with familial lecithin:cholesterol acyltransferase (LCAT) deficiency. (Henry) 24, NATIONAL CHOLESTEROL EDUCATION PROGRAM (ADULT TREATMENT PANEL Ill CLASSIFICATION) TOTAL CHOLESTEROL REFERENCE RANGE Desirable Borderline high High Total cholesterol (mg/dL) <200 7200-239 = 240 HDL CHOLESTEROL REFERENCE RANGE Protective against The higher, Major risk factor for heart disease the better heart disease HDL cholesterol (mg/dL) = 60 40-59 <40Page Is LDL CHOLESTEROL REFERENCE RANGE Optimal Near optimal | Borderline high High Very high LDL _cholesterot <100 100-129 130-159 160-189 >190 (mgidt) TRIGLYCERIDE REFERENCE RANGE Normal Borderline high High Very high Triglyceride (mg/dL) <150 150-199 200-499 2 500 25,_NCEP Guidelines for Acceptable Measurement Error (Henry) ANALYTE TOTAL ERROR BIAS cv Cholesterol <9% 53% 53% Triglyceride 15% <5% <5% HDL-cholesterol 13% <5% 40 Percentage of diabetics <10 % [280% Ketone bodies Usually [Rarely Obestity at onset Rare [Commonly ‘Serum insulin Very tow Normal or high 30. The standard clinical specimen is venous plasma glucose. Glucose is metabolized at foom temperature at a rate of 7 mg/dL/hour (0.4 mmovL/noury; at 4° C, the loss is approximately 2 Mg/AL/hOUT. The rate of metabolism is higher with bacterial contamination or leukocytosis, (Henry) 31. Before an OGTT is performed, individuals should ingest at least 150 giday of carbohydrates for the 3 days preceding the test without limitation in physical activity, and the test should be performed after an overnight 8- to 14-hour fast. The individual should not eat food, drink tea, coffee, or alcohol, or smoke cigarettes during the test, and should be seated. Venous glucose samples are preferably collected in gray-fop tubes containing fluoride and an anticoagulant. (Henry) 32. Whole blood glucose specimens can be analyzed with point-of-care devices. These monitoring devices are used in the home, in the physician's office, or at the bedside in the hospital to monitor for hypoglycemia and hyperglycemia. Most of these devices have been calibrated to give results similar to plasma levels and can report plasma or whole blood readings. Whole blood tends to give approximately 10%-15% lower glucose readings than plasma, but the Percentage varies on the basis of hematocrit, analysis technique, and sample timing (fasting vs. postglucose load). (Henry)Page 16 33. As little as 10% contamination with 5% dextrose will increase glucose in a blood sample by 800 mg/dL or more. (from Dean Rodriquez Clinical Chemistry Review Handbook, 2012) 34. Copper reduction methods for Glucose Estimation Nelson-Somogyi | Glucose method reduces copper in hot alkaline solution to cuprous ion which in tum reduces arsenomolybdic acid in a greenish blue complex Folin-Wu Glucose reduces copper in hot alkaline solution fo Cuprous lon which in tum reduces phosphomolybdic acid forming a blue complex of molybdenum oxide Neocuproine | Cuprous ions were formed by the reduction of cupric with glucose. Neocuproine (2, 9 - dimethyl-1, 10 - method phenanthroline hydrochloride) specifically complexes with cuprous ions to form a yellow color 36. The Michaelis-Menten hypothesis of the relationship between reaction velocity and substrate concentration can be represented mathematically as follows V= Vmax [S]/ Km + [S] where Vis measured velocity of reaction, Vimax is maximum velocity, [S] is substrate concentration, and Km is Michaelis- Menten constant of enzyme for specific substrate, 36. In 1913, Michaelis and Menten hypothesized the role of substrate concentration in formation of the enzyme-—substrate (ES) complex. According to their hypothesis, the substrate readily binds to free enzyme at a low-substrate concentration, ‘With the amount of enzyme exceeding the amount of substrate, the reaction rate steadily increases as more substrate is, added. The reaction is following first-order kinetics because the reaction rate is directly proportional to substrate concentration. Eventually, however, the substrate concentration is high enough to saturate all available ‘enzyme, and the reaction velocity reaches its maximum, When product is formed, the resultant free enzyme immediately ‘combines with excess free substrate. The reaction is in zero-order kinetics, and the reaction rate depends only ‘on enzyme concentration. (Bishop) 37. One of two general methods may be used to measure the extent of an enzymatic reaction: (1) fixed-time and (2) continuous-monitoring or kinetic assay. in the fixed time method, the reactants are combined, the reaction proceeds for a designated time, the reaction is stopped (usually by inactivating the enzyme with a weak acid), and a measurement is made of the amount of reaction that has ‘occurred. The reaction is assumed to be linear over the reaction time; the larger the reaction, the more enzyme is present In continuous-monitoring or kinetic assays, multiple measurements, usually of absorbance change, are made during the reaction, either at specific time intervals (usually every 30 or 60 seconds) or continuously by @ continuous- recording spectrophotometer. 38. ENZYME CLASSES CLASS | CATEGORY TYPE OF REACTION CATALYZED EXAMPLES 1 | Oxidoreductase | Oxidation/reduction reactions Lactate dehydrogenase Glucose-6-phosphate dehydrogenase Glutamate dehydrogenase 2 | Transferase | Transfer of intact group of atoms from one molecule to | Aspartate aminotransferase another ‘Alanine aminotransferase Creatine kinase Gamma glutamyltransferase Gluthione-S-transferase Glycogen phosphorylase Pyruvate kinase 3 | Hydrolase Cleavage of bonds with water Alkaline phosphatase Acid phosphatase Amylase Triacylglycerol lipase Cholinesterase Chymotyrpsin Elastase-1 S-nucleotidase Trypsin 4 | Lyases Cleavage of C-C, C-0, C-Nor other types of bonds; does | Aldolase ‘ot involve water 5 __| Isomerases Convert one isomer to another "Triosephosphate Isomerase 6 | Ligases ‘Bond formation between two groups of atoms; with ATP as | Glutathione synthetase energy source 39, MAJOR ENZYMES OF CLINICAL SIGNIFICANCE ENZYME CLINICAL SIGNIFICANCE 1._Acid phosphatase (ACP) Prostatic carcinoma 2._Alanine aminotransferase (ALT) Hepatic disorder 3._ Aldolase (ALD) ‘Skeletal muscle disorder 4. Alkaline phosphatase (ALP) Hepatic disorder Bone disorderENZYME Page |7 CLINICAL SIGNIFICANCE ‘Amylase (AMS) Angiotensin-converting enzyme (ACE) ‘Aoute pancreatitis, Blood pressure regulation solo ‘Aspartate aminotransferase (AST) Myocardial infarction Hepatic disorder ‘Skeletal muscle disorder '8._Chymotrypsin (CHY) Chronic pancreatitis insufficiency ‘9. Creatine kinase (CK) Myocardial infarction Skeletal muscle disorder 10, Elasiase-1 (E1 Chronic pancreatitis insufficiency 11, Glucose-6-phosphate dehydrogenase (G-6-PD) Drug-induced hemolytic anemia 12, Glutamate dehydrogenase (GL) Hepatic disorder 13, y-Glutamyltransferase (GGT) Hepatic disorder 14, Glutathione-S-transferase (GST) Hepatic disorder 15. Glycogen phosphorylase (GP) ‘Acute myocardial infarction 16. Lactate dehydrogenase (LDH) Myocardial infarction Hepatic disorder Hemolysis Carcinoma 17. Lipase (LPS) ‘Acute pancreatitis, 18, 5-Nucleotidase Hepatic disorder 19, Pseudocholinesterase (PChE) Organophosphate poisoning 20. Pyruvate kinase (PK) Genetic variants Hepatic disorder ‘Suxamethonium sensitivity Hemolytic anemia 21. Trypsin (TRY) ‘Acute pancreatitis 40._CONDITIONS AFFECTING TOTAL LACTATE DEHYDROGENASE PRONOUNCED ELEVATION MODERATE ELEVATION ‘SLIGHT ELEVATION (5. OR MORE TIMES NORMAL) (3-5 TIMES NORMAL) (UP TO 3 TIMES NORMAL) Megaloblastic anemia Widespread carcinomatosis, especially hepatic metastases ‘Systemic shock and hypoxia Hepatitis Renal infarction Myocardial infarction Pulmonary infarction Hemolytic conditions Leukemias Infectious mononucleosis Delirium tremens Muscular dystrophy Most liver diseases Nephrotic syndrome Hypothyroidism Cholangitis CONDITIONS AFFECTING CK ‘PRONOUNCED ELEVATION (5 OR MORE TIMES NORMAL) Duchenne’s muscular dystrophy Polymyositis, Dermatomyositis Myocardial infarction injections Hypothyroidism MILD OR MODERATE ELEVATION (2-4 TIMES NORMAL) ‘Severe exercise, trauma, surgical procedure, intramuscular Delirium tremens, alcoholic myopathy Myocardial infarction, severe ischemic injury Pulmonary infarction Pulmonary edema (some patients) Acute agitated psychoses CONDITIONS AFFECTING AST PRONOUNCED ELEVATION MODERATE ELEVATION SLIGHT ELEVATION (5. OR MORE TIMES NORMAL) (3-5 TIMES NORMAL) (UP TO 3 TIMES NORMAL) ‘Acute hepatocelluar damage Biliary tract obstruction Pericarditis Myocardial infarction Cardiac arryhythmias Cirrhosis Circulatory collapse (shock) Acute pancreatitis Infectious mononucleosis Congestive heart failure Metastatic or primary tumor in liver Muscular dystrophy Pulmonary infarction Delirium tremens Cerebrovascular accident CONDITIONS AFFECTING ALP PRONOUNCED ELEVATION MODERATE ELEVATION ‘SLIGHT ELEVATION (5 OR MORE TIMES NORMAL) (3-5 TIMES NORMAL) (UP TO 3 TIMES NORMAL) Bile duct obstruction (intrahepatic or | Granulomatous or inftrative diseases of | Viral hepatitis extrahepatic) liver Cirrhosis Biliary cirrhosis Osteitis deformans (Paget's disease) Osteogenic sarcoma Hyperparathyroidism Infectious mononucleosis Metastatic tumors in bone Metabolic bone diseases (rickets, osteomalacia) Healing fractures Pregnancy (placental isoenzyme conspicuous) Normal growth pattems in childrenPage |8 41._ CHARACTERISTICS OF ISOENZYMES OF ALKALINE PHOSPHATASE, ‘SOURCE OF ENZYME INHIBITION BY ‘ORDER L-phenylalanine (%) Heat or Urea (%) ANODAL MIGRATION Liver 10 60. 1 Bone 10 90. 2 intestine 75. 60. 4 Placenta 80. 0 3 Regan (carcinoma) 80, 0 3 Measurement of Total ALP Activity REACTION NAME T ‘SUBSTRATE USED ‘COMMENTS ‘Shinowara-Jones-Reinhart | Beta-glycerophosphate Long incubation time; high blank values King-Armstrong | Phenylphosphate Endpoint, requires protein removal Bessey-Lowry-Brock | p-Nitrophenylphosphate Endpoint or kinetic, rapid Bowers-McComb P-Nitrophenyiphosphate Uses phosphate-accepting buffer, reference method 42,_Measurement of Total ACP Activity REACTION NAME ‘SUBSTRATE USED ‘COMMENTS Bodansky Beta-glycerophosphate Lengthy assay, nonspecific Gutman, King-Armstrong Phenylphosphate Nonspecific Hudson p-Nitrophenylphosphate Rapid, nonspecific ‘Babson and Reed Alpha-naphthylphosphate Complicated, less sensitive Roy [Thymolphthalein monophosphate | More specific for prostatic form Rietz, Guilbault 4-Methylumbeliferonephosphate Fluorescent, some improved sensitivity 43. Interferences with the assay of total acid phosphatase: A variety of factors produce low levels of acid phosphatase. fluoride inhibits the enzyme. Selection of a proper anticoagulant is important, since both oxalate and heparin have been shown to produce decreased activity. Storage conditions are critical since changes in pH and prolonged storage at room temperature both result in loss of enzyme activity ‘The major factor producing flase elevations is hemolysis. Since the erythrocytes contain significant amounts of acid phosphatase, loss of enzyme from these cells strongly influences the value obtained from a serum or plasma sample. Failure to use an anticoagulant, results in release of enzyme from platelets, contributing to an increase in the amount of enzyme measured.in methodologies that measure product formation at 410 nm or near this wavelength), hemoglobin and bilirubin in high concentrations contribute to the absorbance and yield falsely elevated enzyme values. (Calbreath) 44, ENZYMES IN CARDIAC DISORDERS AMI occurs when there is abrupt reduction in blood flow to a region of myocardial tissue, usually caused by atherosclerosis of the coronary arteries. Enzyme | Onset of Elevation (h) T Duration of Elevation (days) cK 48 34 KM | 48 23 ‘AST 8-12 5 LD 12:24 10 45. Demonstration of elevated levels of CK-MB, greater than or equal to 6% of the total CK, is considered a good indicator of myocardial damage, particularly AMI, Other nonenzyme proteins, called troponins, have been found to be even more specific and may elevate in the absence of CK-MB elevations. Following myocardial infarction, the CK-MB levels begin to rise within 4 to 8 hours, peak at 12 to 24 hours, and retum to normal levels within 48 to 72 hours. @ishop) 46. CK: Tanzer-Gilvarg method assesses the rate of the forward reaction in which creatine is converted to creatine phosphate, 47. CK: Oliver-Rosalki, the reverse reaction in which creatine is produced from creatine phosphate. 48. LD: The Wacker Method (forward) for quantitation of LD activity utilizes the lactate -» pyruvate reaction with th formation of NADH from NAD, The 340 nm absorbance can be read directly, allowing kinetic assays to be performed 49. LD: The Wroblewski-LaDue method employs the reverse reaction: pyruvate > lactate. In this situation NADH serves as a cosubstrate and is consumed during the course of the reaction. If kinetic measurement activity is carried out, a decrease in 340 nm absorbance is observed.Page 19 50. ISOENZYMES OF LACTATE DEHYDROGENASE. ISOENZYME CHAIN ‘APPROXIMATE % OF TOTAL NORMALLY TISSUE RICH IN THE COMPOSITION PRESENT IN SERUM ISOENZYME UD; HHHH 29-37 Heart, brain, erythrocytes LD» HHHM 42-48 Heart, brain, erythrocytes LD: HHNIM 16-20 Brain, kidne} LD. HMMM. 2-4 Liver, skeletal muscle, kidney LDs MMMM 05-15 Liver, skeletal muscle, ileum 51. Routine measurement of electrolytes usually involves only Na’, K°, CI, and HCO; (as total CO,). These values may be used to approximate the anion gap (AG), which is the difference between unmeasured anions and unmeasured cations, (Bishop) 52. There are two commonly used methods for calculating the anion gap. The first equation is AG = Na’ - (CI + HCOs) ‘The reference range for the AG using this calculation is 7-16 mmol/L. The second calculation method is AG = (Na’+ K’) - (CI' + HCO) Ithas a reference range of 10-20 mmol. An elevated anion gap may be caused by uremia/renal failure, which leads to PO, and SO,” retention; ketoacidosis, as Seen in cases of starvation or diabetes, methanol, ethandl ‘ethylene. glycol potsoning, or salicylate; lactic. acidosis, hypernatremia; and instrument error. Low anion gap values are rare but may be seen with hypoalbuminemia (decrease in unmeasured anions) or severe hypercalcemia (increase in unmeasured cations. (Bishop) 53. All sodium ions must be neutralized by counter-ions, most of which, in blood, are constituted by chloride and bicarbonate ions, and, to a lesser degree, by phosphate, sulfate, and protein carboxylate groups. Normal serum sodium is about 140 MEQ/L, chloride is usually around 100 mEgiL, and bicarbonate around 2 mEq/L. The anion gap is then defined as Na+ ={Cl- + HCO5-), which for normal individuals is around 16. This 16 mEq/L really accounts for the other counter-ions that neutralize sodium but are not measured in serum. 54. Renin-angiotensin system (RAS): hormones, renin, angiotensin, and aldosterone work together to regulate blood pressure. A sustained fall in blood pressure causes the kidney to release renin. This is converted to angiotensin in the circulation. Angiotensin then raises blood pressure directly by arteriolar constriction and stimulates the suprarenal glands to produce aldosterone that promotes sodium and water retention by kidney, such that blood volume and blood pressure increase. 55. About 70% of T4 is bound to thyroxine-binding globulin (TBG), 20% to transthyretin (formerty called binding prealbumin), and 10% to albumin. (Henry) 56, LABORATORY VALUES IN HYPOTHYROIDISM AND HYPERTHYROIDISM LABORATORY VALUES IN TABORATORY VALUES IN HYPOTHYROIDISM. HYPERTHYROIDISM. T4, total Decreased T4, total Increased T4, free Decreased T4, free Increased 73, direct Decreased 3, direct Increased 3 uptake Decreased TS uptake Increased TSG Normal TEC Normal or decreased TSH Increased TSH Low-normal or undetectable 57. MAJOR ENDOGRINE GLANDS AND THEIR HORMONES. ENDOCRINE HORMONE FUNCTION HYPOSECRETION | _HYPERSECRETION GLAND “Anterior Pituitary | Growth hormone (GH) _| Major effects are Hyposecretion during | Hypersecretion produces: Gland ‘Somatotropin directed to the growth | childhood leads to ‘| gigantism (in childhood) (adenohypophysis) | Most abundant of skeletal muscles —_| pituitary dwarfism | and acromegaly (in hormone of the and long bones of the adulthood) anterior pituitary bod Prolactin (PRL) ‘Stimulates production of breast milk. ‘Adrenocorticotropic | Stimulates adrenal hormone (ACTH) cortex to release its hormones. Thyrolropic hormone | Stimulates the thyroid (TH) gland to release ‘Thyroid-stimulating thyroid hormones hormoneENDOCRINE GLAND HORMONE FUNCTION HYPOSECRETION Page |10 HYPERSECRETION Anterior Pituitary Gland (adenohypophysis) Follcie-stimulating hormone (FSH) Luteinizing hormone aH Beginning at puberty, stimulates follicle development and estrogen production by female ovaries; promotes sperm | production in males ‘Beginning at puberty, stimulates ovulation, converts the ruptured ovarian follicle to a corpus luteum, and causes the corpus luteum to produce progesterone; stimulates male testes to produce testosterone ‘Sterility in both male and female Sterility in both male and female Posterior Pituitary Gland (neurohypophysis) Oxytocin Released in significant amounts only during childbirth and in nursing women ‘Stimulates powerful uterine contractions and causes milk ejection in nursing woman ‘Antidiuretic hormone (ADH) Vasopressin ‘Causes kidney tubule cells to reabsor and conserve body water and increases blood pressure by constricting arterioles Diabetes insipidus Thyroid Gland Parathyroid Glands Thyroxine (12) Triiodothyronine ( Body's metabolic hormone. It increases the rate at which cells oxidize glucose and is necessary for normal growth and development. Hyposecretion of thyroxine results in cretinism in children Hypersecretion results from Grave's disease and other forms of hyperthyroidism Calcitonin | Parathyroid hormone (PTH) ‘Causes calcium to be deposited in long | bones ‘Causes bone calcium to be liberated to blood Hyposecretion results in tetany Hypersecretion leads to extreme bone wasting and fractures ‘Adrenal Cortex Adrenal Cortex Mineralocorticoids mainly aldosterone (outermost) Glucocorticoids which include cortisone and cortisol (Middle) ‘Sex hommones - androgens (male) with ‘some estrogen (female) Regulate sodium and potassium ion reabsorption by the kidneys. Their release is primaniy stimulated by low Na’ high K” | levels in the blood. Enable the body to resist long-term stress by increasing blood glucose levels and decreasing the inflammatory response, ‘A generalized hypoactivity of adrenal cortex leads to Addison's disease Hypersecretion of adrenal cortex hormones can result in hyperaldosteronism, Cushing's disease, and/or masculinization (innermost) ‘Adrenal Medulla | Catecholamines: Hypersecretion leads to Epinephrine symptoms typical of (adrenaline) ‘symphathetic nervous Norepinephrine activity (noradrenaline)Page |12 ENDOCRINE HORMONE FUNCTION HYPOSECRETION | HYPERSECRETION GLAND Islets of Tasulin Tnoreases the rate of | Diabetes melitus Langerhans of the glucose uptake and Pancreas by beta cells metabolism by body cells ‘Glucagon ‘Stimulates the liver to release glucose to by alpha cells blood, thus increasing blood glucose levels ‘Ovaries Estrogen ‘Stimulates the Hyposecretion ‘maturation of the hampers the ability of female reproductive | a woman to conceive organs and and bear children development of secondary sex characteristics of the female; in cooperation with progesterone, it causes the menstrual cycle Progesterone Ttworks with estrogen | Hyposecretion in establishing the hampers the ability of ‘menstrual cycle ‘a woman to conceive and bear children Testes Testosterone Promotes maturation | In cases of of the male hyposecretion, the reproductive organs, | man becomes sterile male secondary sex _| (sterility) characteristics, and production of sperm by testes Pineal Gland Melatonin Affects biological rhythms and reproductive behavior Thymus gland Thymosin ‘Cause the maturation of T lymphocytes 58. Anatomically, the adrenal is divided into two distinct parts: The medulla (inner layer) and the cortex (outer layer). The medulla, which is of neural crest origin (ectoderm), stores and secretes catecholamines. The cortex is of mesenchymal origin and is further divided into three zones: The outermost zona glomerulosa, which produces mineralocorticoids; the zona fasciculata, which is responsible for glucocorticoid production; and the inner zona reticularis, which ‘synthesizes androgens. (Henry) 59. Estrogens are responsible for growth of the uterus, fallopian tubes, and vagina, promotion of breast development, maturation of the external genitalia, deposition of body fat into the female distribution, and termination of linear growth. Estradiol is the most potent of the estrogens. 60._DISEASE STATE HORMONE LEVEL Male Primary deficiency __Klinefelter’s syndrome High High Low 5 ‘Secondary deficiency Panhypopituitarism Low Low Low S Primary excess Testicular tumor Low Low High e Secondary excess Precocious puberty High High High = Other ‘Seminiferous tubule failure High Normal ‘Normal = Other Parlial androgen insensitivity Normal High High = Female Primary deficiency _ Menopause High High = Low Secondary deficiency Sheehan's syndrome Low Low = Low [Classification Example FSH LH Testosterone Estradiol | Primary excess Feminizing ovarian tumor Low Low Ee High Secondary excess Gonadotropin-producing tumor (rare) High High = High Other Polyoystic ovarian syndrome _ Normal High High = Other Masculinizing ovarian tumor Low Low High =Page |12 61. In some individuals, high levels of blood cholesterol or triglycerides are caused by genetic abnormalities in which either too much is synthesized or too little is removed. High levels of cholesterol and/or triglycerides in most people, however, are a result of increased consumption of foods rich in fat and cholesterol, smoking, and lack of exercise or a result of other disorders or disease states that affect lipid metabolism, such as diabetes, hypertension, hypothyroidism, obesity, liver and kidney diseases, and alcoholism. 62. Henderson-Hasselbalch equation: equation that mathematically describes the dissociation characteristics of weak acids and bases and the effect on pH; pH = pKa (6.1) + log of the ratio of bicarbonate to carbon dioxide (HCOs/H:CO3). @ishop) 63. Potentiometry is the measurement of electrical potential due to the activity of free ions - change in volatage indicates activity of each analyte. Uses: pH and pCOs tests. (from Dean Rodriquez Clinical Chemistry Review Handbook, 2012) 64. Amperometry is the measurement of the current flow produces by an oxidation reaction. Uses: pO», glucose, chloride ‘and peroxidase determinations. (from Dean Rodriquez Clinical Chemistry Review Handbook, 2012) 65. For each degree of fever in the patient, pO» will fall 7% and pCO, will rise 3%. (from Dean Rodriquez Clinical Chemistry Review Handbook, 2012) 66. DRUGS OF ABUSE 1. Opiates © Chemical modification of natural products yields heroin and hydrocodone © Fully synthetic opiods are meperidine (Demerol) and methadone 2. Amphetamines 3. Cocaine © Derived from the leaves of coca plant (Genus Erythroxylon) 4. Cannabinoids Marijuana from the flowers of the hemp plant Hashish from the resin of the hemp 5. Phencyclidine (PCP, angel dust) and lysergic acid diethylamide (LSD) 6. Ethyl alcohol (ethanol, grain alcohol) ‘Most commonly abused substance in the US, and probably in the entire World INFLUENCE OF ACUTE ETHANOL INGESTION ON ETHANOL LEVELS AND BEHAVIOR STAGES OF IMPAIRMENT BY ETHANOL, BLOOD ALCOHOL ‘SIGNS AND SYMPTOMS, (% wiv) 0.01 — 0.05 No obvious impairment, some changes observable on performance testing 0.03 - 0.12 Mild euphoria, decreased inhibitions, some impairment of motor skills 0.09 0.25 Decreased inhibitions, loss of eritical judgment, memory impairment, diminished reaction time 0.18 0.30 Mental confusion, dizziness, strongly impaired motor skills (staggering, slurred speech) 0.27 0.40 Unable to stand or walk, vomiting, impaired consciousness 0.35- 0.50 Coma and possible death ‘Whiskey Blood Concentration Tnfluence (Ounces) 1-2 10 - 50 mg/dL (2.2 - 10.9 mmol/L) | None to mild euphoria 34 50 - 100 mg/dL (10.9 - 21.7 mmol/L) Mild influence on stereoscopic vision and dark adaptation or greater 100 mg/dL. (21.7 mmol/L) Legally intoxicated 46 100 - 150 mg/dL (21.7 - 32.6 mmol/L) Euphoria; disappearance of inhibition; prolonged reaction time 67 150 - 200 mg/dL (32.6 - 43.4 mmol/L) Moderately severe poisoning; reaction time greatly prolonged: loss of inhibition and slight disturbances | in equilibrium and coordination e9 200 - 250 mg/dL (43.4 - 84.3 mmol/L) ‘Severe degree of poisoning; disturbances of equilibrium and coordination; retardation of the thought processes and clouding of consciousness 10-15 | 250 - 400 mg/d (64.3 - 86.8 mmol/L) Deep, possibly fatal comaPage [43 POISONING 1. GASES ‘© Carbon monoxide ~ results from incomplete combustion of carbon-containing material in fires, gasoline engines and cigarette smoke (mtd: spectrophotometry and co-oximetry) © Cyanide — in the form of hydrocyanic acid (HEN, prussic acid), used as rodentcide and insecticide; odor of bitter almonds (mid: spectrophotometry) 2. HEAVY METALS ‘© Methods for determination: Atomic absorption spectrophotometry Anodic stripping voltametry Inductively coupled plasma mass spectrometry Reinsch test (antimony, arsenic, bismuth, mercury and selenium) © Iron = from iron-containing tablet and solution; children prone to accidental ingestion of large amounts of iron resulting in toxicity © Lead ~ present in paints, gasoline (formerly), and storage batteries, some eating utensils, plates and ceramics, drinking water form lead pipes © Arsenic — in insecticides, pesticides and herbicides; high affinity for keratin ‘© Cadmium ~ ingestion of acidic foods stored or prepared in metal containers composed or lined with cadmium; industrial exposure © Mercury ~ used in industry and farming © Aluminum ~ abundant in Earth's crust and is widely present in the environment; aluminum toxicity has been noted in patients who are receive long-term hemodialysis 3. BROMIDE ‘© Once widely used as an analgesic but it has been removed because of its toxicities ‘© Measured in the serum by spectrophotometric methods 67. Cocaine’s short halflife is a result of rapid hepatic hydrolysis to inactive metabolites. This is the major route of elimination. Only a small portion of the parent drug can be found in urine after an administered dose, The primary product of hepatic metabolism is benzoylecgonine, which is primarily eliminated in urine. The half-life of benzoylecgonine is 4~7 hours. The presence of this metabolite in urine is a sensitive and specific indicator of cocaine use. It can be detected in urine for up to 3 days after a single use. In chronic heavy abusers, it can be detected in urine for up to 20 days after the last dose. The primary screening procedure for identification of cocaine use is detection of benzoylecgonine in urine by immunoassay. Confirmation testing is done by GC/MS. (Bishop) 68. Cannabinoids are a group of psychoactive compounds found in marijuana. Of these, THC is the most potent and abundant. Marijuana, or its processed product, hashish, can be smoked or ingested. (Bishop) 69. The most specific and sensitive method for drug screening is the coupling of gas chromatography to mass spectrometry. (Calbreath) 70. THERAPEUTIC DRUGS ‘CARDIOTROPICS Most commonly used to treat congestive hear failure and Digitalis glycosides: digoxin and digitoxin Procainamide (Pronestyl) cardiac arrythmias Quinidine Lidocaine (Xylocaine) Propranolol Disopyramide “ANTICONVULSANTS Phenobarbital Used in the treatment of seizure disorders, in particular grand ‘mal, petit mal, and psychomotor seizures and other generalized seizure disorders such as tic douloreux (trigeminal neuralgia) Phenytoin (Ditantin) Primidone (Mysoline) Ethosuximide (Zarontin) Carbamazepine (Tegretol) Valproic acid (Depakene) “ANTIASTHMATICS. ‘ANTIINFLAMMATORY DRUGS Theophylline Most commonly prescribed anti-asthmatic drug Bronchodilator for the treatment of moderate to severe asthma, both for the prevention of attacks and for treatment of symptomatic exacerbations. ‘Acetaminophen (Tylenol) Acetyisalicylic acid IMMUNOSUPPRESSIVES Cyclosporine Prednisone Cyclophosphamide (Cytoxan) ‘CHEMOTHERAPEUTIC AGENTS: Methothrexate An anti-neoplastic agent Important immunosuppressive agent used in the treatment of psoriasis, rheumatoid arthritis, and some collagen vascular diseasesPage |14 DRUGS FOR TREATMENT OF MANIC-DEPRESSION Lithium — anti-manic agent and are used for the prophylaxis Used in the treatment of psychiatric affective disorders, and treatment of bipolar disorder (manic-depressive psychosis) Tricyclic Antidepressants: Amitriptyline, Imipramine, Nortriptyline, Desipramine, Doxepin 71. BENZODIAZEPINES: Among this group of drugs, the most prominent is VALIUM; they are used therapeutically, as so- called minor tranquilizers. 72. ASPIRIN (acetylsalicylic acid) is a commonly used analgesic, antipyretic, and anti-inflammatory drug. Several immunoassay methods are available; the most common is a chromogenic assay known as the TRINDER reaction, which reacts salicylate with ferric nitrate to form a colored complex that is then evaluated spectrophotometrically. 73. ACETAMINOPHEN, either solely or in combination with other compounds, is a commonly used analgesic drug. In healthy subjects, therapeutic dosages have few adverse effects. Overdose of acetaminophen, however, is associated with a severe HEPATOTOXICITY. 74. The goal of drug administration is to achieve the therapeutic range, that level of concentration in the bloodstream which provides the optimum amount of medication for treatment of the clinical disorder. A blood level of medication below the therapeutic range is considered subtherapeutic, meaning it provides no clinical benefit. (Calbreath) 75. TUMOR MARKERS (from Dean Rodriquez Clinical Chemistry Review Handbook, 2012) TUMOR MARKERS ‘ASSOCIATED CANCERS: ‘AFP Hepatic and testicular cancers ALP (placental ALP) ‘Lung cancer ‘Amylase Pancreatic cancer BRCA1 Breast or ovarian cancer CA-125 ‘Ovarian cancer (treatment and recurrence) CA-15.3 Breast cancer ((reatment and recurrence) CA-19.9 Gastric, pancreatic and colorectal cancers CA-50 Gastric and pancreatic cancers (treatment and recurrence) (CA-27.20 Breast cancer (treatment and recurrence) Calcitonin Medullary thyroid canoer Cathepsin-D Breast cancer CEA Colorectal, stomach, breast, lung cancer (Weatment and recurrence) a ‘Small cell lung cancer, prostate cancer Estrogen receptor (ER) Breast cancer ‘GGT Hepatoma HER-2mneu Breast cancer (efficiency of frastuzumab or herceptin therapy) Nuclear matrix protein (NMP) Urinary bladder cancer From Dean Rodiiquez Cinical Chemistry Review Fandbook 2072 76. Capillary blood is the preferred specimen for some tests, such as newborn screening tests. (Bishop 77. Delta check: an algorithm in which the most recent result of a patient is compared with the previously determined value, 78. MULTI-RULE PROCEDURES: The “multirule” procedure was developed by Westgard and Groth to further judge whether control results indicate out-of-control situations, 125 One control observation exceeding the mean + 2s. A warning rule that initiates testing of control data by other rules, 13s One control observation exceeding the mean + 3s. Allows high sensitivity to random error. 22s Two control observations consecutively exceeding the same + 2s or - 2s. Allows high sensitivity to systematic error. Ris One control exceeding the + 2s and another exceeding the - 2s. Allows detection of random error 41 Four consecutive control observations exceeding + 1s or 1s, Ths allows the detection of systematic error. 10, Ten consecutive control observations falling on one side or the other of the mean (no requirement for SD size). This allows the detection of systematic error. 79. RANDOM ERROR is one with no trend or means of predicting it, Random errors include such situations as mislabelling a sample, pipeting errors, improper mixing of sample and reagent, voltage fluctuations not compensated for by instrument circuitry, and temperature fluctuations. Violations of the 1(28), 1(38) and R(4S) Westgard rules are usually associated with random error. To assess the situation, the sample is assayed using the same reagents. If a random error occurred, the same mistake may not be made again, and the result will be within appropriate control limits. (Calbreath)Page [45 80. A SYSTEMATIC ERROR, on the other hand, will be seen as a trend in the data. Control values gradually rise (or fall) from the previously established limits. This type of error includes improper calibration, deterioration of reagents, sample instability, instrument drift, or changes in standard materials. All the Westgard rules that indicate trends identify systematic errors. 2(28), 4(1S) and 10(x) rule, If reassay does not correct the problem by bringing the control values within the + 2SD range, further analysis of the data is necessary. A stepwise evaluation of the procedure needs to be carried out to determine where the problem lies. This examination could include preparing new control materials, restandardizing the assay, checking wavelength or other instrument settings, or making new reagents, The past history of the specific test may be helpful in deciding which steps take first. Reagents that are close to their expiration date should be discarded and remade. The laboratory records for calibration and the calibration schedule may point to a need for a new standard curve. The process should proceed in a logical fashion to identify and correct the problem. Adequate records should be kept of each step. Good documentation will permit easier correction of the problem in the future. (Calbreath) 81. TREND: values for the control that continue to either increase or decrease over a period of 6 consecutive days (PER) 82. Trend is formed by control values that either increase or decrease for six consecutive days. Main cause is deterioration reagents. (from Dean Rodriquez Clinical Chemistry Review Handbook, 2012) 83. SHIFT: 6 or more consecutive daily values that distribute themselves on one side of the mean value line, but maintain a constant level (e.g, an increase shift) might be due to deterioration of a standard but is remedied by preparation of a new standard. (PER) 84. Shift is formed by control values that distribute themselves on one side or either side of the mean for six consecutive days. Shift in the reference range is due to transient instrument differences. Main cause is improper calibration of instrument. (from Dean Rodriquez Clinical Chemistry Review Handbook, 2012) 85, Outliers are control values that are far from the main set of values. They are highly deviating values and are caused by random or systematic error. (from Dean Rodriquez Clinical Chemistry Review Handbook, 2012) 88. VARIABLES: Statistical questions are often posed in terms of input versus output, cause and effect, or correlation between two or more variables. The input or cause is considered an independent variable because it is already determined and so is not influenced by other factors. Examples of independent variables are age, gender, temperature, and time. In contrast, dependent variables are those things that might change in response to the independent variable. Examples of dependent variables are blood glucose concentration, enzyme activities, and the presence or absence of malignancy. For graphical display, the INDEPENDENT VARIABLE IS PLOTTED ALONG THE HORIZONTAL (X) AXIS OR ABSCISSA, WHILE DEPENDENT VARIABLES ARE PLOTTED ALONG THE VERTICAL (Y) AXIS OR ORDINATE. (Henry) 87. The t test, also called the paired t test or the student t test compares the accuracy of two methods in that it tests the difference between the mean value of each procedure. The reference or current method is considered to reflect the true value. The t test is based on a null hypothesis, which assumes that there is no difference between the two methods. (Brown) 88. The t test is used to determine wheter there is a statiscally siginificant difference between the means of two groups of data. (Rodriguez) 89. The F test is used to compare the precision of two procedures. (Brown) 90. F test is used to determine whether there is statistically significant difference between the standard deviations of two groups. (Rodriguez) 91. Until this epidemic of overweight/obesity lessens, approximately two of every three individuals measured may be either ‘overweight (BMI 25-29.9 kg/m’) or obese (BMI 230 kg/m’) by National Heart, Lung, and Blood Institute (NHLBI) standards, On the other side of the spectrum is the individual who may be malnourished/undemourished and possibly also underweight (BMI < 18.5 kg/m’) 92. Diurnal variation may be encountered when testing for hormones, iron, acid phosphatase, and urinary excretion of most electrolytes such as sodium, potassium and phosphate. (Henry) 93. Incorrect application of the tourniquet and fist exercise can result in erroneous test results, Using a tourniquet to collect blood to determine lactate concentration may result in falsely increased values. Prolonged tourniquet application may also increase serum enzymes, proteins, and protein-bound substances, including cholesterol, calcium, and triglycerides, due to hemoconcentration. (Henry) 94, Basal state: early morning before the patient has eaten or become physically active. This is a good time to draw blood specimens because the body is at rest and food has not been ingested during the night. Bishop) 95. If blood pressure cuff is used as a tourniquet, itis inflated 60 mmHg. (from Dean Rodriquez Clinical Chemistry Review Handbook, 2012)96, Page |16 COMPARISON OF BIOLOGIC SAFETY CABINETS CABINETS APPLICATIONS TYPE FACE RADIONUCLEOTIDES/ BIOSAFETY PRODUCT VeLocrTy AIRFLOW PATTERN TOXIC CHEMICALS LEVEL(S) PROTECTION remy Glass 1" open front_75 ‘In at front; rear and top No 23 No through HEPA filter ClassiTypeA 75 70% Recirculated through No 23 Yes HEPA; exhaust through HEPA Type BY 700 30% Recireulated through Yes 23 Yes HEPA; exhaust via HEPA and (low levels/ hard-ducted volatility) Type 82 700 No recirculation, total exhaust Yes 23 Yes via HEPA and hard-ducted Type 83 700 Same as IIA, but plenums under Yes 23 Yes negative pressure to room land exhaust air is ducted Class NA Supply air inlets Yes 34 through 2 HEPA fiers 97. 98, 99, BASIC APPROACHES TO AUTOMATION: There are three basic approaches with instruments: CONTINUOUS FLOW, CENTRIFUGAL ANALYSIS, AND DISCRETE ANALYSIS. All three can use batch analysis (.e., large number of specimens in one run), but only discrete analyzers offer random access, or stat, capabilities. (Bishop) POINT-OF-CARE APPLICATIONS. a, POC glucose is the highest-volume POC test in most health care institutions. b. Several different manufacturers offer instrumentation designed to measure POC chemistries (most frequently electrolytes) and/or blood gases, c. The most common POC coagulation test is activated clotting time (ACT). d. At the present time, only minimal hematology POCT has been available. In past years, the spun hematocrit was the ‘most common POC hematology test. , Connectivity has been the most significant recent development in POCT. Connecti document testing. (Bishop) y Is the ability to electronically ‘When a fire is discovered, all employees are expected to take the actions in the acronym RACE: (Strasinger) a. Rescue—rescue anyone in immediate danger b. Alarm—activate the institutional fire alarm system ¢. Contain—close all doors to potentially affected areas 4d. Extinguish—attempt to extinguish the fire, if possible; exit the area 100._ FIRE EXTINGUISHER "Type of Extinguisher A Pressurized water/Dry chemical B Dry chemical/Carbon dioxide G Carbon dioxide/Halon/Dry chemical D Metal X/Special dry chemical 101. Pressurized-water extinguishers, as well as foam and multipurpose dry-chemical types, are used for Class A fires. 102 103. Multipurpose dry-chemical and carbon dioxide extinguishers are used for Class B and C fires. Halogenated hydrocarbon extinguishers are particularly recommended for use with computer equipment. Class D fires present special problems, and extinguishment is left to trained firefighters using special dry-chemical extinguishers. Analytic chemicals exist in varying grades of purity: analytic reagent (AR); ultrapure, chemically pure (CP); United States Pharmacopeia (USP); National Formulary (NF); and technical or commercial grade. (Bishop) Specifications set by CAP define three grades of water: a. Type | reagent water: for procedures that require maximum water purity: preparation of standard solutions, ultramicrochemical analyses, measurements at nanogram or subnanogram concentrations, and tissue or cell culture methods b. Type Il reagent water: for most laboratory determinations in chemistry, hematology, microbiology, immunology and other clinical laboratory areas c. Type Ill reagent water: for most qualitative measurements/examinations; most procedures in urinalysis, parasitology and histology, washing glasswaresPage |47 104. Types of Centrifuge Horizontal or swinging bucket __| Allow the tubes to attain a horizontal position in the centrifuge when spinning and centrifuge a vertical position when the head is not moving ‘The specimen cups in the horizontal centrifuge heads are in a vertical position when the centrifuge is at rest. During centrifugation, the cups move to a horizontal position. As the specimen is centrifuged, the particles being sedimented travel down through the liquid to the bottom of the tube. When the centrifuge stops and the tubes swing to a vertical position there may be remixing of the sediment with the supernatant liquid, These centrifuge heads are capable of speeds up to about 3000 RPM. Higher speeds than this will generally cauase excessive heat buildup as a result of air friction. Fixed-angle or angle-head Have angled compartments for the tubes and allow small particles to sediment centrifuge more rapidly ‘Angle centrifuge heads are capable of higher speed and contain driled holes that hold the tubes at a fixed angle (approximately 52° angle with the center shaft around which they rotate). There is much less heat developed during centrifugation because of very low air friction. During centrifugation, the particles travel across the column of liquid to the side of the tube where they clump together and then rapidly move to the bottom of the tube. Ultracentrifuges: High-speed centrifuges used to Separate layers of different specific gravities, They are commonly used to separate lipoproteins, The chamber is usually refrigerated to counter heat produced through friction, 105. TEMPERATURE CONVERSIONS (Bishop) Centigrade (°C) to Kelvin °K} *K="0+273 Gentigrade (°C) to Fahrenheit °F) oF = ("Ox 18) #32 Fehrenhet (°F to Centigrade (°C) $C =F - 32)x 0556 CLINICAL MICROSCOPY 106. PREGNANCY TEST: BIOLOGIC TESTS TEST ‘ANIMAL USED MODE OF POSITIVE RESULT INJECTION 1. Ascheim-Zondek | Immature ferale mice Subcutaneous | Formation of hemorthagic follicles and corpora lutea 2. Friedman Mature virgin female rabbit Marginal ear vein | Hyperemic uterus and corpora hemorthagica 3. Hogben | Female toad (enopus aevis) South Afican | Lymph sac | Oogenesis| clawed frog - carries eggs throughout the year 4 Gallc-Mainini | Male fog (Rana pipiens or Rana clamitans, | Subcutaneous | Spermalogenesis leopard or grass frog); Male toad (Bufo bufo or B. americanus) 5._Frank-Berman | Immature female rats Subcutaneous | Ovarian hyperemia 6._Kupperman Female rat Intraperitoneal | Ovarian hyperemia 107. The first clinically bioassay for HCG was introduced by Ascheim (1927) and Zondek (1931) and was characterized by enlargement and luteinisation of the corpus luteam of the immature mouse after injection of urine from normally pregnant women. Zondek noted similar results when the urine from women with choriocarcinoma or ovarian cancer or from men with testicular neoplasms was used. These assays were followed by Friedman's test and the Xenopus laevis test, using urine from pregnant women, with the end point being ovulation in the rabbit and South African toad, respectively. ‘Two subsequent tests reported in 1948 - the Rana pipiens frog test and the Galli-Mainini toad test - measured the release of spermatozoa from the male frog and toad, respectively, two to four hours after injection of urine from pregnant women. (Henry 19” Ed.) 108. Membrane cassette tests for pregnancy determination are one-step solid-phase enzyme immunoassays designed to detect the presence of hCG in urine or serum. hCG is a hormone secreted by the trophoblast of the developing embryo; it rapidly increases in the urine or serum during the early stages of pregnancy. In a normal pregnancy, hCG can be detected in serum as early as 7 days following conception, and the concentration doubles every 1.3 to 2 days. It is subsequently ‘excreted into the urine. Levels of hCG reach a peak of approximately 200,000 miUimL. at the end of the first trimester. Because the test cassette contains all necessary reagents, this is called immunochromatography. The test band region is precoated with anti-alpha hCG antibody to trap hCG as it moves through the membrane caused by capillary action. When the patient specimen is added, it reconstitutes an antibeta hCG antibody, which is complexed to colloidal gold particles. This complex is trapped by the anti-alpha hCG and forms a colored complex in the test region. This may be in the form of a straight line or a plus sign. A positive test results if a minimum concentration of approximately 25 mIU/mL is present. The control region contains a second antibody directed against the anti-beta hCG antibody. This second antibody reacts with the excess anti-beta hCG antibody gold particles to indicate that the test is working correctly. (Stevens)Page [18 109. Calculated Glomerular Filtration Estimates: Formulas have been developed to provide estimates of the GFR based on the serum creatinine without the urine creatinine. These formulas are becoming more frequently used in clinical medicine. As discussed, the creatinine clearance is not useful in detecting early renal disease. Therefore the calculated clearances are being used for monitoring patients already diagnosed with renal disease or at risk for renal disease. In addition, the formulas are valuable when medications that require adequate renal clearance need to be prescribed. The most frequently used formula was developed by COCKCROFT AND GAULT. It is also used to document eligibility for reimbursement by the Medicare End Stage Renal Disease Program and for evaluating patient placement on kidney transplant lists. Variables included in the original formula are age, sex, and body weight in kilograms, a (140 - age)Qweight in kilograms) ‘= 72> serum creatinine im mg/d ‘The results are multiplied by 0.85 for female patients, Modifications to the original formula substitute ideal body weight in kilograms and adjusted body weight in kilograms. This is done to correct for weight that may not be the result of muscle mass, i., fatty tissue. The calculation for ideal body weight (IBW) is: ‘Males: 50 kg + 2.3 kg for each inch of height over 60 inches Females: 45.5 kg + 2.3 kg for each inch of height over 60 inches. ‘The calculation for adjusted body weight (AjBW) is: LBW + 0.3 CABW>ABW) ‘A newer formula, called the Modification of Diet in Renal Disease (MDRD) system, ultiizes additional variables and does not include body weight. The variables include ethnicity, blood urea nitrogen, and serum albumin. Several variations of the formula are available, ullizing one or more of the additional variables. An example of the MDRD study formula is: GFR— 170 serum creatinine °999 x age°176 >< 0.822 Gif patient is female) < 1.1880 ( if patient is black) >< BUN°?7° > serum albumin 19315 A laboratory advantage of this formula is that, as body weight is omitted, all results are available from the laboratory ‘computer information, and the calculation can be performed automatically by the instrument performing the serum creatinine. 110. By far the greatest source of error in any clearance procedure utilizing urine is the use of improperly timed urine ‘specimens. (Strasinger) 111. Clearance of inulin, a complex polysaccharide produced by certain plants, has been widely regarded as the gold standard for measuring GFR. (Henry) 112. Urine drug specimen: The collector adds bluing agent (dye) to the toilet water reservoir to prevent an adulterated specimen. The collector checks the urine for abnormal color and for the required amount (30-45 mL). The collector checks that the temperature strip on the specimen cup reads 32.8C-37.7C. (Strasinger) 113. Containers for routine urinalysis should have a wide mouth to facilitate collections from female patients and a wide, flat bottom to prevent overturning. They should be made of a clear material to allow for determination of color and clarity The recommended capacity of the container is 50 mL, which allows 12 mL of specimen needed for microscopic analysis, additional specimen for repeat analysis, and enough room for the specimen to be mixed by swirling the container. (Strasinger) 114. In routine urinalysis, clarity is determined in the same manner that ancient physicians used: by visually examining the mixed specimen while holding it in front of a light source. The specimen should, of course, be in a clear container. (Strasinger) Clear — transparent, no visible particulates Hazy — few particulates, print easily seen through urine Cloudy —_many particulates, print blurred through urine Turbid — print cannot be seen through urine Milky ~ may precipiate or clot 115, LABORATORY CORRELATIONS IN URINE TURBIDITY Acidic urine ‘Amorphous urates, radiographic contrast media Alkaline urine "Amorphous phosphates, carbonates ‘Soluble with heat “Amorphous urates, uric acid crystals ‘Soluble in dilute acetic acid RBCs, amorphous phosphates, carbonates Insoluble in dilute acetic acid WECs, bacteria, yeast, spermatozoa ‘Soluble in ether Lipids, lymphatic fluid, chyle 116. _ A major disadvantage of using a urinometer to measure specific gravity is that it requires a large volume (10 to 15 mL) of specimen. (Strasinger) 117. Calibration of the refractometer is performed using distilled water that should read 1.000. If necessary, the instrument contains a zero set screw to adjust the distilled water reading. The calibration is further checked using 5% NaCl, which as shown in the refractometer conversion tables should read 1,022 + 0.001, or 9% sucrose that should read 1.034 + 0.001. (Strasinger)Page |19 118. Equipment found in the urinalysis laboratory commonly includes refrigerators, centrifuges, microscopes, and water baths. Temperatures of refrigerators and water baths should be taken daily and recorded. Calibration of centrifuges is customarily performed every 3 months, and the appropriate relative centrifugal force for each setting is recorded, Centrifuges are routinely disinfected on a weekly basis. Microscopes should be kept clean at all times and have an annual professional cleaning, (Strasinger) 119.__ TUBULAR REABSORTPTION [ ‘SUBSTANCE LOCATION ACTIVE TRANSPORT Glucose, amino acids, salts Proximal convoluted tubule ‘Movement ofa substance across cell Chloride ‘Ascending loop of Henle membranes to te beecteam by Sodium Proximal and distal convoluted tubules PASSIVE TRANSPORT Water Proximal convoluted tubule, descending ‘Movement of molecules across @ membrane loop of Henle, and collecting duct by diftusion because of a physical gradient Urea Proximal convoluted tubule and ascending loop of Henle ‘Sodium ‘Ascending loop of Henle 120. Many medications, including rifampin, phenolphthalein, phenindione, and phenothiazines, produce red urine. (Strasinger) 121. _ Normal urine produces only a small amount of rapidly disappearing foam when shaken, and a large amount of white foam indicates an increased concentration of protein. (Strasinger) 122, __ URINE pH ACID URINE ALKALINE URINE a ipararaton peed Vomiting Dehydration Renal tubular acidosis pera ht a cacrohette Fyetoncecfssse podcing bcteta erseast eat rearen oan on Vegetarian ai Cranberry juice Old specimens Medications (methenamine mandeate[Mandetamine,fosfomycin ‘romethamine) 123.__URINE ODOR ‘Aromatic Normal Foul, ammoniacike Bacterial decomposition, UTI Fruity, sweet Ketones (DM, starvation, vomiting) Maple syrup Maple syrup urine disease Mousy Phenylketonuria Rancid Tyrosinemia ‘Sweaty feet Isovaleric academia Cabbage Methionine malabsorption. Bleach Contamination 124. __ SUMMARY OF CHEMICAL TESTING BY REAGENT STRIP Test | _ Principle | Reagent Strip Reaction Glucose | Double sequential | Reagent strip manufacturers use several diferent chromogens, including potassium [enzyme reaction _| iodide (green to brown) and tetramethylbenzidine (yellow to green) Bilirubin | Diazo reaction Bilirubin combines with 2,4-dichloroaniline diazonium salt or 2,6-dichlorobenzene- diazonium tetrafluoroborate in an acid medium to produce an azodye, with colors | | ranging from increasing degrees of tan or pink to violet, respectively Ketones | Sodium ‘Acetoacetic acid in an alkaline medium reacts with sodium nitroprusside fo produce a nitroprusside purple color. The test does not measure beta-hydroxybutyric acid and is only slightly reaction Sensitive to acetone when glycine is also present; Specific | pKa change of ‘As the specific gravity increases, the indicator changes from blue (7,000 (alkaline), gravity Polyelectrolyte through shades of green, to yellow (1.030 [acid)). pH Double indicator in the pH range 5 to 9 measured by the reagent sirips, one sees colors progressing system from orange at pH 5 through yellow and green to a final deep blue at pH 9. Protein | Protein error of ‘At a pH level of 3, both indicators appear yellow in the absence of protein; however, indicators as the pro tein concentration increases, the color progresses through various shades of green and finally to blue Blood Pseudoperoxidase | In the presence of free hemoglobin’ myoglobin, uniform color ranging from a negative activity of hemoglobin | yellow through green to a strongly positive green-blue appears on the pad. in contrast, intact red blood cells are lysed when they come in contact with the pad, and the liberated hemoglobin produces an isolated reaction that results in a speckled pattern on the pad.