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.Page |20 Test | Principle I Reagent Strip Reaction Urobilinogen | Ehrich’ reaction The reagent strip reactions for urobilinogen differ between Muitistix and Chemstrip much more significantly than do other reagent strip parameters. Multistix uses Ehvlich’s aldehyde reaction, in which urobilinogen react with p- dimethylaminobenzaldehyde (Ehriich reagent) to produce colors ranging from light to dark pink. Chemstrip incorporates an azo-coupling (diazo) reaction using 4 methoxybenzene diazoniumtetrafiuoroborate to react with urobilinogen, producing colors ranging from white to pink, This reaction is more specific for urobilinogenthan the Ehrlich reaction, Nitrite Greiss reaction Nitrite at_an acidic pH reacts with an aromatic amine (para-arsanilic acid or sulfanilamide) to form a diazonium compound that then reacts with telrahydrobenzoquinolin compounds to produce a pink colored azodye. Leukocytes | Esterase reaction The aromatic compound then combines with a diazonium salt present on the pad to produce a purple azodye. 125. Automated reagent strip readers use a spectropholtometric measurement of light reflection termed reflectance photometry, Reflectance photometry uses the principle that light reflection from the test pads decreases in proportion to the intensity of color produced by the concentration of the test substance. (Strasinger) 126. _ Reagent strips are packaged in opaque containers with a desiccant to protect them from light and moisture. Strips are removed just prior to testing, and the bottle is tightly resealed immediately. Bottles should not be opened in the presence of volatile fumes. Manufacturers recommend that reagent strips be stored at room temperature below 30 °C. (Strasinger) 127. _ Reporting SSA turbidity: (Strasinger) Grade Turbidity Protein range (mg/dl) Negative No increase in turbidity <6 Trace Noticeable turbidity 6-30 1+ Distinct turbidity with no granulation 30-100 2+ Turbidity with granulation, no flocculation 100-200 3+ ‘Turbidity with granulation and flocculation 200-400 a CClumps of protein >400 128. Prior to the development of current reagent strip methods that are specific for albumin, detection of microalbuminuria required collection of a 24-hr urine specimen. Specimens were tested using quantitative procedures for albumin. Results were reported in mg of albumin/24 hours or as the albumin excretion (AER) in yg/min, With these methods, microalbumin is considered significant when 30 to 300 mg of albumin is excreted in 24 hours or the AER is 20-200 g/min. 129.__ MICROSCOPIC TECHNIQUES Technique Function Bright-field Used for routine urinalysis microscopy Phase-contrast Enhances visualization of elements with low refractive indices, such as hyaline casts, mixed microscopy cellular casts, mucous threads and Trichomonas Polarizing microscopy | Aids in identification of cholesterol in oval fat bodies, fatty casts, and crystals Dark-field microscopy | Aids in identification of Treponema pallidum Fluorescence Allows visualization of naturally fluorescent microorganisms or those stained by a fluorescent dye microscopy Interference-contrast_| Produces a three-dimensional microscopy-image and layer-by-layer imaging of a specimen 130. Two types of interference-contrast microscopy are available: modulation contrast (Hoffman) and differential interference contrast (Nomarski). (Strasinger) 131. The first procedure to standardize the quantitation of formed elements in the urine microscopic analysis was developed by Addis in 1926. The Addis count, as it is called, used a hemocytometer to count the number of RBCs, WECs, casts, and epithelial cells present in a 12-hour specimen, Normal values have a wide range and are approximately 0 to 500,000 RBCs, 0 to 1,800,000 WECs and epithelial cells, and 0 to 5000 hyaline casts.(Strasinger) 132. _ Lipid stains: Oil Red O and Sudan Ill: Stains triglycerides and neutral fats orange-red. (Strasinger) 133. Squamous cells are the largest cells found in the urine sediment. They contain abundant, irregular cytoplasm and a prominent nucleus about the size of a RBC. They are often the first structures observed when the sediment is examined under low-power magnification. (Strasinger) 134. A variation of the squamous epithelial cell is the clue cell, which does have pathologic significance. Clue cells are indicative of vaginal infection by the bacterium Gardnerella vaginalis. (Strasinger) 135. _ RTE cells are the most clinically significant of the epithelial cells. The presence of increased amounts is indicative of necrosis of the renal tubules, with the possibilty of affecting overall renal function. (Strasinger)Page [2a 136. Conditions producing tubular necrosis include exposure to heavy metals, drug-induced toxicity, hemoglobin and myoglobin toxicity, viral infections (hepatitis B), pyelonephritis, allergic reactions, malignant infiltrations, salicylate Poisoning, and acute allogenic transplant rejection. (Strasinger) 197. _In cases of acute tubular necrosis, RTE cells containing large, nonlipid-filed vacuoles may be seen along with normal renal tubular cells and oval fat bodies. Referred to as “bubble cells," they appear to represent injured cells in which the ‘endoplasmic reticulum has dilated prior to cell death. (Strasinger) 138. The presence of urinary eosinophils is primarily associated with drug-induced interstitial nephritis; however, small numbers of eosinophils may be seen with urinary tract infection (UTI) and renal transplant rejection. Evaluation of concentrated, stained urine sediment is required for performing a urinary eosinophil test. Sediment may be concentrated by routine centrifugation alone or with cytocentrifugation, The preferred eosinophil stain is Hansel; however, Wright's stain ‘ean also be used. Eosinophils are not normally seen in the urine; therefore, the finding of more than 1% eosinophils is considered significant. (Strasinger) 139. Formation of casts at the junction of the ascending loop of Henle and the distal convoluted tubule may produce structures with a tapered end. These have been referred to as cylindroids, but they have the same significance as casts. (Strasinger) 140. Epithelial cell casts may be difficult to differentiate from WBCs, particularty if degeneration has occurred. Staining and the use of phase microscopy can be helpful to enhance the nuclear detail needed for identification 141. Uric acid crystals occur at low pH (5-5.5) and are seen in a variety of shapes, including rhombic or four-sided flat plates, prisms, oval forms with pointed ends (lemon-shaped), wedges, rosettes, and irregular plates. Most are colored, typically yellow or reddish brown. Rarely, they are colorless and hexagonal, resembling cystine. Unlike cystine, they show birefringence with polarized light. (Henry) 142, In the presence of severe liver disorders, three rarely seen crystals may be found in the urine sediment. They are crystals of tyrosine, leucine, and bilirubin. (Strasinger) 143. Sulfonamide (Sulfadiazine) Crystals. These crystals may be seen in urine of acid pH and may take on various morphologies, depending on the form of drug involved. They may be seen as yellow-brown sheaves of wheat with central bindings, striated sheaves with eccentric bindings, rosettes, arrowheads, petals, needles, and round forms with radial striations. They are occasionally coloriess. Confirmatory testing is by the diazo reaction. High-performance liquid chromatographic and colorimetric methods have also been described. With the advent of soluble sulfonamides, sulfa crystals are not as frequently found in urine, especially when urine is examined at 37° C, Before this development, these crystals could be seen in the urine of patients on sulfonamide therapy who were inadequately hydrated. This could result in renal tubular damage if crystal formation occurred within the nephron, Currently, sulfamethoxazole is seen with some regulanty, 144. Precipitation of antibiotics is not frequently encountered except for the rare observation of ampicilin crystals following massive doses of this penicillin compound without adequate hydration. Ampicillin crystals appear as colorless needles that tend to form bundles following refrigeration. (Strasinger) 145. The finding of clumps of calcium oxalate crystals in fresh urine may be related to the formation of renal calcull, because the majority of renal calculi are composed of calcium oxalate. (Strasinger) 148. Calculi may be of various sizes, commonly described as sand, gravel, or stone, The physical characteristics of the various calculi rarely will suffice for their identification, but a few points are worth noting. Uric acid and urate stones are typically yellow to brownish red and are moderately hard. Phosphate stones are usually pale and friable. Calcium oxalate stones are very hard, often of a dark color, and typically have a rough surface. Cystine stones are yellow-brown and feel somewhat greasy. (Henry) 147. Magnesium ammonium phosphate (struvite) forms stones at alkaline pH, where the ammonium level is high. These tend to form in the pelvis of the kidney but apparently are not attached to papillae, as are the calcium stones. They may, however, develop on preexisting nuclei when infection from organisms such as Proteus causes alkalization of the urine. Struvite stones may become large, forming casts of the kidney pelvis and showing staghoms. (Henry) 148. Triple phosphate (ammonium magnesium phosphate) crystals are commonly seen in alkaline urine. In their routine form, they are easily identified by their prism shape that frequently resembles a ‘coffin lid". As they disintegrate, the crystals may develop a feathery appearance. Triple phosphate crystals are birefringent under polarized. (Strasinger) 149. Triple phosphate (ammonium magnesium phosphate) crystals are one of the most easily identified urine crystals, although they commonly shaw variation in size. They are colorless, three to six-sided prisms with oblique ends referred to as coffin lids. They may form colorless sheets or flakes. (Henry) 150. The most frequent parasite encountered in the urine is Trichomonas vaginal. (Strasinger) 151. Fecal contamination of a urine specimen can also result in the presence of ova from intestinal parasites in the urine sediment. The most common contaminant is ova from the pinworm Enferobius vermicularis. (Strasinger) 152. The most frequently used screening tests are the acid-albumin and cetyitrimethylammonium bromide (CTAB) turbidity tests and the metachromatic staining spot tests, In the acid-albumin and the CTAB tests, a thick, white turbidity formsPage |22 when these reagents are added to urine that contains mucopolysaccharides. Turbidity is usually graded on a scale of 0 to 4 after 30 minutes with acid-albumin and after § minutes with CTAB, Metachromatic staining procedures use basic dyes to react with the acidic mucopolysaccharides. (Strasinger) 153. WEGENER’'S GRANULOMATOSIS causes a granuloma-producing inflammation of the small blood vessels of primarily the kidney and respiratory system. Key to the diagnosis of Wegener's granulomatosis is the demonstration of ANTINEUTROPHILIC CYTOPLASMIC ANTIBODY (ANCA) in the patient's serum. 154. Patients with serotonin-producing carcinoid tumors often have marked ten-fold increases in urinary §-HIAA excretion. Ingestion of serotonin-rich foods, such as bananas, chocolate, plums, or walnuts, or medications containing guaifenesin May produce false-positive elevations (Henry) 155. Inbom error of metabolism: Testing for many substances is now performed using tandem mass spectrophotometry (MS/MS). MS/MS is capable of screening the infant blood sample for specific substances associated with particular IEMs, (Strasinger) 156. The most well known of the aminoacidurias, phenylketonuria (PKU) is estimated to occur in 1 of every 10,000 to 20,000 births and, if undetected, results in severe mental retardation, Interruption of the pathway also produces children with fair complexions— even in dark-skinned families—owing to the decreased production of tyrosine and its pigmentation metabolite melanin, (Strasinger) 157. Melanin reacts with ferric chloride, sodium nitroprusside (nitroferricyanide), and Ehrlich reagent. In the ferric chloride tube test, a gray or black precipitate forms in the presence of melanin and is easily differentiated from the reactions produced by other amino acid products. The sodium nitroprusside test provides an additional screening test for melanin. A red color is produced by the reaction of melanin and sodium nitroprusside. Interference due to a red color from acetone and creatinine can be avoided by adding glacial acetic acid, which causes melanin to revert to a green-black color, whereas acetone tums purple, and creatinine becomes amber (Strasinger) 158. Fanconi syndrome Etiology Inherited in association with cystinosis and Harinup disease or acquired through exposure to toxic agents Primary Urinalysis, Glucosuria, possible cystine crystals Results Other Significant Tests | Serum and urine electrolytes, amino acid chromatography 159. FERRIC CHLORIDE TUBE TEST Phenylketonuria Blue-green Tyrosyluria Transient green Alkaptonuria Transient blue Melanuria Gray-black Maple syrup urine disease Green-gray Indicanuria Violet-blue with chloroform 5-HIAA, Blue-green 160. CHEMICAL TESTS ‘Qualitative Tests for Protein Heller's Robert's White ring at the zone of conatct Spiegler’s Biuret Violet for albu Rose for albuminoses and peptones Heal and acetic acid ‘SSA Purdy’s White turbidity/cloudiness, Potassium ferrocyanide Picric acid ‘Quantitative Tests for Prote Esbach's 24° ~ read height of coagulum Kwileck's| 72°C for 5 minutes ~ read height of coagulum Tsuchiya’s ‘Same as Esbach’s Kingsbury-Clark Degree of turbidity is measured by comparison with standard turbidiies Biuret Uses the same principle as that used for serum protein which depends upon the presence of peptide linkages in protein ‘Sugars, Benedict's Reducing substances Green-orange-red ‘Osazone or Phenylhydrazine Glucose, fructose, lactose & pentose (Kowarsky) Crystalline needles Nylander’s Glucose & other reducing subs Brown to black colorPage |23 Moore Heller Glucose & other reducing subs Canary yellow to black Borcharat’s Fructose Seliwanoff Red color Resorcinol-HC1 Rubner's Lactose: Brick red color wired ppt Glucose: Red color wiyellow ppt Bial Orenal Green soln Tauber’ Chey red Ketones Frommer's Acetone Purpish re ring Rothera’s ‘Acetone & aceloacelic aid Rose of purple ring Tange ‘Acetone & acetoacelic add Purple ring ‘ectest ‘Acetone & acetoacetic acid Ketostix Purple color Gerhards ‘cetoacelic acid Bordeaux red color BILE PIGMENTS (Bilirubin, urobilinogen & urobilin) Gmelin Bile pigments Play of colors Sith Bile pigments Emerald green Harrison's spot Bile pigments Blue to green color Tetotest Bile pigments Blue to purple mat Wallace and Diamond Urobilinogen Cherry red color Schiesinger Urobitn Greenish fuorescence HEMOGLOBIN Benzidine Green-blue Guiac Blue Onttorudine Blue MELANIN Screening est Urine will urn brown to black ‘Thormahlen (Fresh urine) Dark green or blue color Blackberg & Wanger (24-hr urine) | Brown to black ppt CHLORIDE Fantus Reddish ppt Mercurimeti tration Blue-violet colored complex Schales & Schales CALCIUM Sulkowitch [Precipitation 161. Cerebrospinal fluid: Specimens are collected in three sterile tubes, which are labeled 1, 2, and 3 in the order in which they are withdrawn. Tube 1 ‘Chemical and serologic tests Tube 2 Tubes Gell count 162. Cerebrospinal fluid: Transferrin is the major beta globulin present; also, a separate carbohydrate-deficient transferrin fraction, referred to as “tau,” is seen in CSF and not in serum. (Strasinger) 163. _ DIFFERENTIAL DIAGNOSIS OF BLOODY CSF ‘A traumatic tap occurs in about 20% of lumbar punctures. Distinction of a traumatic puncture from a pathologic hemorthage is, therefore, of vital importance. Although the presence of crenated RBCs is not useful, the following observations may be helpful in distinguishing the two forms of bleeding ‘A. Ina traumatic tap, the hemorrhagic fluid usually clears between the first and third collected tubes but remains relatively uniform in subarachnoid hemorrhage. B. Xanthochromia, microscopic evidence of erythrophagocytosis, or hemosiderinaden macrophages indicate a ‘subarachnoid bleed in the absence of a prior traumatic tap. RBC lysis begins as early as 1~2 hours after a traumatic, tap. Thus, rapid evaluation is necessary to avoid false-positive resultsPage [24 C. A commercially available latex agglutination immunoassay test for cross-linked fibrin derivative d-dimer is specific for fibrin degradation and is negative in traumatic taps. However, false-positive results might be expected in disseminated intravascular coagulation, fibrinolysis, or trauma from repeated lumbar punctures. 164. _Xanthochromic CSF: RBCs must usually remain in the CSF for approximately 2 hours before noticeable hemolysis begins; therefore, a xanthochromic supematant would be the result of blood that has been present longer than that introduced by the traumatictap. Care should be taken, however, to consider this examination in conjunction with those previously discussed, because a very recent hemorthage would produce a clear supematant, and introduction of serum protein from a traumatic tap could also cause the fluid to appear xanthochromic. To examine a bloody fluid for the Presence of xanthochromia, the fluid should be centrifuged in a microhematocrit tube and the supernatant examined against a white background. (Strasinger) 165. Clear CSF specimens may be counted undiluted, provided no overlapping of cells is seen during the microscopic examination. When dilutions are required, calibrated automatic pipettes, not mouth pipetting, are used. Dilutions for total cell counts are made with normal saline, mixed by inversion, and loaded into the hemocytometer with a Pasteur pipette. (Strasinger) CSF DILUTIONS (Strasinger 3") CLARITY DILUTION Clear specimens May be counted undiluted ‘Slightly hazy 1:10 Hazy 1:20 Slightly cloudy 1:100 ‘Cloudy 1:200 Slightly bloody Bloody 7:10.00 Turbid 166. Lysis of RBCs must be obtained prior to performing the WBC count on either diluted or undiluted CSF specimens. Specimens requiring dilution can be diluted in the manner described previously, substituting 3% glacial acetic acid to lyse the RBCs. Addition of methylene blue to the diluting fluid stains the WBCs, providing better differentiation between neutrophils and mononuclear cells. (Strasinger) 167. During the first trimester, the approximately 35 mL of amniotic fluid is derived primarily from the maternal circulation. During the latter third to half of pregnancy, the fetus secretes a volume of lung liquid necessary to expand the lungs with growth. During each episode of fetal breathing movement, secreted lung liquid enters the amniotic fluid, as evidenced by lung surfactants that serve as an index of fetal lung maturity. After the first trimester, fetal urine is the major contributor to the amniotic fluid volume. At the time that fetal urine production occurs, fetal swallowing of the amniotic fluid begins and regulates the increase in fluid from the fetal urine. (Strasinger) Failure of the fetus to begin swallowing results in excessive accumulation of amniotic fluid (polyhydramnios) and is an indication of fetal distress, often associated with neural tube disorders. Polyhydramnios may be secondarily associated with fetal structural anomalies, cardiac arrhythmias, congenital infections, or chromosomal abnormalities. Increased fetal swallowing, urinary tract deformities, and membrane leakage are possible causes of decreased amniotic fluid (cligohydramnios). Oligohydramnios may be associated with umbilical cord compression, resulting in decelerated heart rate and fetal death, (Strasinger) 168. Amniotic Fluid Color Color Significance Colorless Normal Blood-streaked Traumatic tap, abdominal trauma, intra-amniotic hemorrhage Yellow Hemolytic disease of the newborn (bilirubin) Dark green Meconium Dark red-brown Death 169. Amniotic fluid for fetal lung maturity (FLM) tests should be placed in ice for delivery to the laboratory and refrigerated up to 72 hours prior to testing or kept frozen and tested within 72 hours. Frozen specimens should be thoroughly mixed, by vortexing, after thawing. Repeated freeze-thawing is not recommended. Specimens for cytogenetic studies are maintained at room temperature or body temperature (37 °C incubation) prior to analysis to prolong the life of the cells needed for analysis. (Strasinger) 170. Chromosome abnormalities, such as trisomy 21 (Down syndrome). (Strasinger) 171. Amniotic Fluid Test T Normal Values at Term ‘Significance Bilirubin scan ‘Awa > 0.025 Hemolylic disease of the newborn Aipha-fetoprotein 2.0 MoM ‘Neural ube disorders Lecithin-sphingomyelin ratio 2.0 Fetal lang maturity “Amniostat-fetal lung maturity Positive Fetal lung maturity phosphotidylalycerol Foam stabiity index SAT Fetal lung maturityPage [25 Test I Normal Values at Term | Significance Microviscosity [355mg9 Fetal lung maturity Optical density 650 nm [20.150 Fetal lung maturity Lamellar body count 232,000/uL_ Fetal lung maturity 172. Incteased levels of alpha-fetoprotein (AFP) in both the matemal circulation and the amniotic Nuid can be indicative of fetal neural tube defects, such as anencephaly and spina bifida, Elevated amniotic fluid AFP levels are followed by measurement of amniotic acetyicholinesterase (ACHE). The test is more specific for neural tube disorders than AFP, provided its not performed on a bloody specimen, because blood contains ACHE. (Strasinger) 173. Peritoneal fluid. Psammoma bodies containing concentric striations of collagen-like material can be seen in benign conditions and are also associated with ovarian and thyroid malignancies, (Strasinger) 174. The gastric juice is obtaind by the insertion of a gastric tube into the stomach. The Rehfuss tube is introduced through the mouth, The Levin tube is another type which is inserted through the nose. Disposable plastic tubes are usually employed, 175. Normal gastric fluid is pale gray and contains mucus. In the fasting state, food particles should NOT be present as this would indicate an incomplete digestion process. The presence of large amounts of bile producing a yellow-green color and the appearance of blood should be reported. (Strasinger 3° ed.) 176. Gastric fluid. Fasting specimen may vary from a few mL. to 50 mL with an average of 30 mL. Amounts over 50 mL are regarded as abnormal. Following the Ewald's test meal, the amount is expected to be between 20 - 60 mL, sometimes up to 120 mL. After an alcohol test meal, or histamine stimulation, the total volume of accretion varies from 45 to 150 mL, 177. VARICOCELE is the most common cause of male infertility. It is the hardening of veins that drain the testes. This causes blood from the adrenal vein to flow into the spermatic vein. (Hart) 178. The normal sperm has an oval-shaped head approximately Sum long and 3 wm wide and a long, flagellar tail approximately 45 um long. Critical to ovum penetration is the enzyme-containing acrosomal cap located at the tip of the head. The acrosomal cap should encompass approximately half of the head and covers approximately two thirds ofthe sperm nucleus. The neckpiece attaches the head to the tail and the midpiece. The midpiece is the thickest part of the tail because it is surrounded by a mitochondrial sheath that produces the energy required by the tail for motility. (Strasinger) 179. The normal semen specimen should be easily drawn into a pipette and form droplets that do not appear clumped or stringy when discharged from the pipette, Normal droplets form a thin thread when released from the pipette, Droplets with threads longer than 2 centimeters are considered highly viscous. Ratings of 0 (watery) to 4 (gel-like) can be assigned to the viscosity report. Viscosity can also be reported as low, normal, and high. Increased viscosity and incomplete liquefaction impede sperm motility. (Strasinger) 180. Assessment of sperm motility should be performed on well mixed, liquefied semen within 1 hour of specimen collection. The practice of examining sperm motilty at timed intervals over an extended period has been shown to serve no useful purpose. To provide Ocontinuity in reporting, laboratories should place a consistent amount of semen under the same size coverslip, such as 10 yl under a 22 x 22 mm coverslip. The percentage of sperm showing actual forward movement can then be estimated after evaluating approximately 20 high-power fields. (Strasinger) 181. Sperm Motility Grading Grade WHO Criteria 40 a Rapid, straight-line motility 3.0 b Slower speed, some lateral movement 2.0 b ‘Slow forward progression, noticeable lateral movement 1.0 ce No forward progression oO d No movement 182. Sperm concentration. The most commonly used dilution for is 1:20 prepared using a mechanical (positive-displacement) pipette. Dilution of the semen is essential because it immobilizes the sperm prior to counting. The traditional diluting fluid contains sodium bicarbonate and formalin, which immobilize and preserve the cells; however, good results can also be achieved using saline and distilled water. 183. Low sperm concentration may be caused by lack of the support medium produced in the seminal vesicles. This can be indicated by a low to absent fructose level in the semen, Specimens can be screened for the presence of fructose using the resorcinol test that produces an orange color when fructose is present. A normal quantitative level of fructose is equal to or greater than 13 umol per ejaculate. This can be determined using spectrophotometric methods. Specimens for fructose levels should be tested within 2 hours or frozen to prevent fructolysis. (Strasinger) 184, Additional chemical testing performed on semen may include determination of the levels of neutral a-glucosidase, zinc, citric acid, and prostatic acid phosphatase. Just as decreased fructose levels are associated with a lack of seminal fluid:Page |26 decreased neutral a-glucosidase suggests a disorder of the epididymis. Decreased zinc, citrate, and acid phosphatise indicate a lack of prostatic fluid. (Strasinger) 185. Synovial fluid is collected by needle aspiration called arthrocentesis. The amount of fluid present varies with the size of the joint and the extent of fluid buildup in the joint. For example, the normal amount of fluid in the adult knee cavity is less than 3.5 mL, but can increase to greater than 25 mL with inflammation. (Strasinger) 186. Synovial fluid: When sufficient fluid is collected, it should be distributed into the following tubes based on the required tests: a. sterile heparinized tube for Gram stain and culture b. Aheparin or EDTA tube for cell counts c. Anonanticoagulated tube for other tests d. A sodium fluoride tube for glucose analysis Powdered anticoagulants should not be used because they may produce artifacts that interfere with crystal analysis. 187. The total leukocyte count is the most frequently performed cell count on synovial fluid. Red blood cell (RBC) counts are seldom requested. To prevent cellular disintegration, counts should be performed as soon as possible or the specimen should be refrigerated. Very viscous fluid may need to be pretreated by adding a pinch of hyaluronidase to 0.5 mL of fluid or one drop of 0.05% hyaluronidase in phosphate buffer per milliliter of fluid and incubating at 37C for minutes. (Strasinger) 188. Synovial fluid: Traditional WBC diluting fluid cannot be used because it contains acetic acid that causes the formation of mucin clots, Normal saline can be used as a diluent, If it is necessary to lyse the RBCs, hypotonic saline (0.3%) or saline that contains saponin is a suitable diluent. Methylene blue added to the normal saline stains the WBC nucle, permitting separation of the RBCs and WECs during counts performed on mixed specimens, 189. Serous fluids (3P's): An ethylenediaminetetraacetic acid (EDTA) tube is used for cell counts and the differential. Sterile heparinized evacuated tubes are used for microbiology and cytology. For better recovery of microorganisms and abnormal cells, concentration of large amounts of fluid is performed by centrifugation, Chemistry tests can be run on clotted specimens in plain tubes or on heparinized tubes. Specimens for pH must be maintained anaerobically in ice. (Strasinger) 190. Pleural fluid should be collected in three sterile tubes that are labeled sequentially, The first tube is used for culture and Gram stain and the others for cell counts, Wright's stain, total protein, glucose, cytology and other studies as indicated. 191. At least three tubes of pericardial fluid should be obtained: an EDTA tube for gross and microscopic examination, a sterile plain or heparinised tube for microbiological studies, and a heparinised tube for chemical analyses. 192. For the collection of peritoneal fluid, three collection tubes should be used. For cell counts, gross observations and Wright’s stained smear, an EDTA tube should be used. A sterile heparinised tube can be used for microbiological examination, and a heparinised tube for chemistry determinations. 193. Pale stool associated with biliary obstruction and steatorrhea appears bulky and frothy and frequently has a foul odor. ‘Stools may appear greasy and may float. (Strasinger) 194. SPUTUM Color Colorless or translucent: made up of mucus only White or yellow: pus is present Gray: pus, epithelial cells are present Bright green or greenish: presence of bile, infection by Pseudomonas aeruginosa Red or bright red: fresh blood or hemorrhage: TB, bronchiectasis Anchovy sauce or rusty brown: old blood, pneumonia Prune juice: pneumonia and chronic cancer of the lungs Olive greenigrass green: cancer Black: inhalation of dust or dir, carbon, charcoal, anthracosis and heavy smokers. 195. SPUTUM ODOR: Foul and putrid in lung gangrene and in advanced necrotizing tumors due to decomposition by anaerobic bacteria; sweetish in bronchiectasis, TB, with cavities etc. Freshly expectorated sputum has no odor. Old sputums undergo putrefaction rapidly. 196. Dittrich plugs are yellowish or gray caseous matter, the size of a pinhead or navy bean which when crushed will give a foul odor. They come from the bronchi and bronchioles. They may occur in chronic bronchitis and bronchial asthma, also in healthy persons. They were formerly regarded as a sign of TB, 197. Myelin globules in sputum. Myelin globules have litle or no significance but may be mistaken for Blastomyces (yeast-like fungi). They are colorless, round , oval or pea-shaped of various sizes. 198. Leukocytes, primarily neutrophils, are seen in the feces in conditions that affect the intestinal mucosa, such as ulcerative colitis and bacterial dysentery. Microscopic screening is performed as a preliminary test to determine whether diarrhea is being caused by invasive bacterial pathogens including Salmonella, Shigella, Campylobacter, Yersinia, and enteroinvasive E.coli. Bacteria that cause diarrhea by toxin production, such as Staphylococcus aureus and Vibrio spp., viruses, and parasites usually do not cause the appearance of fecal leukocytes. (Strasinger)Page |27 199. Fecal leukocytes: In an examination of preparations under high power, as few as three neutrophils per high-power field can be indicative of an invasive condition. (Strasinger) 200. Quantitative fecal fat analysis is used as a confirmatory test for steatorrhea. Analysis requires the collection of at least a 3-day specimen (Strasinger) 201. FECAL OCCULT BLOOD TESTING: In addition to sample handling and testing, diet is very important in compromising these tests. The high rate of false positives is due to the lack of specificity of the reagents and the presence of peroxidase activity in other fecal constituents, as well as the sensitivity of the different chromogens to peroxidase activity. The myoglobin and hemoglobin of ingested meat and fish have peroxidase activity that may falsely indicate the presence of occult blood. Bacteria in the bowel, as well as ingested vegetables and fruits, such as horseradish, ‘turnips, bananas, black grapes, pears, and plums, have peroxidase and can falsely elevate fecal peroxidase activity. False-negative tests occur in the presence of large amounts of vitamin G and other antioxidants. 202. APT test: The material to be tested is emulsified in water to release hemoglobin and, after centrifugation, 1% sodium hydroxide is added to the pink hemoglobincontaining supernatant. In the presence of alkali-resistant fetal hemoglobin, the solution remains pink (Hb F), whereas denaturation of the maternal hemoglobin (Hb A) produces a yellow-brown supernatant after standing for 2 minutes. (Strasinger) 203. Normal stool pH is between 7 and 8; however, increased use of carbohydrates by intestinal bacterial fermentation increases the lactic acid level and lowers the pH to below 5.5 in cases of carbohydrate disorders. (Strasinger) 204. Quality control and QA programs are part of the institutional CQ! and total quality management (TQM). Whereas QA is designed to maintain an established level of quality, TQM and CQI are designed to develop methods to continually improve the quality of healthcare. (Strasinger 4" ed., page 118) 205. The most widely used plan for quality improvement in health care is the Plan-Do-Check-Act (PDCA) strategy also known as the Plan-Do-Study-Act (PDSA) cycle. (Strasinger) 208. Turnaround time refers to time from ordering a test through analysis in the laboratory to the charting of the report (Strasingen) 207. CORRECT HANDWASHING TECHNIQUE: Wet hands with warm water. Apply antimicrobial soap. Rub to form lather, create friction, and loosen debris. Thoroughly clean between fingers, including thumbs, under fingernails and rings, and up to the wrist, for at least 15 SECONDS. Rinse hands in a DOWNWARD POSITION. Dry with a paper towel ‘Tum off faucets with a clean paper towel to prevent recontamination, Noo ReNe 208. The health-care setting provides abundant sources of potentially harmful microorganisms. These microorganisms are frequently present in the specimens received in the clinical laboratory. Understanding how microorganisms are transmitted (chain of infection) is essential to preventing infection. The chain of infection requires a continuous link between a source, a method of transmission, and a susceptible host, The source is the location of potentially harmful microorganisms, such as a contaminated clinical specimen or an infected patient. Microorganisms from the source are transmitted to the host. This may occur by direct contact (e.g., the host touches the patient, specimen, or a contaminate object), inhalation of infected material (e.g., aerosol droplets from a patient or an uncapped centrifuge tube), ingestion of a contaminated substance (e.g., food, water, specimen), or from an animal or insect vector bite. Once the chain of infection is complete, the infected host then becomes another source able to transmit the microorganisms to others. (Strasinger) 209. Hazardous chemicals should be labeled with a description of their particular hazard, such as poisonous, corrosive, or carcinogenic. The National Fire Protection Association (NFPA) has developed the Standard System for the Identification of the Fire Hazards of Materials, NFPA 704.7 This symbol system is used to inform fire fighters of the hazards they may encounter with fires in a particular area. The diamond-shaped, color-coded symbol contains information relating to health, flammability, reactivity, and personal protection/special precautions. Each category is graded on a scale of 0 to 4, based on the extent of concern. These symbols are placed on doors, cabinets, and containers. BLUE I RED T YELLOW WHITE | HEALTH HAZARD | FIRE HAZARD REACTIVITY ‘SPECIFIC HAZARD 4 Deadly Flash Point 4 May deteriorate OXY Oxidizer 3 Extreme danger 4 Below 73 F 3 Shock and heat may deteriorate ACID Acid 2 hazardous 3 Below 100 F 2 Violent chemical change ALK Alkali 4 Slightly hazardous 2 Below 200 F 41 Unstable if heated COR Corrosive © Normal material 4 Above 200 F 0 Stable USE NO WATER O Will not burn RADIATION.Page |28 HEMATOLOGY 210. Order of Draw: Evacuated Tube and Syringe (Henry) Blood-culture tubes (yellow) ‘Sodium-citrate tube (blue stopper) ‘Serum tubes with or without clot activator or gel separator Heparin tubes with or without gel (green stopper) EDTA tubes (lavender stopper) Glycolytic inhibitor tubes (gray stopper) 211. Venipuncture Procedure (Rodak) ‘The recommended order of draw is as follows: Blood culture or sterile tubes (ie., yellow stopper) 2. Coagulation tube (i.., ight-blue stopper) 3. Serum tube with or without clot activator or gel (Le, red, gold, or red-gray marbled stopper) 4. Heparin tube (ie. green or light-green stopper) 5. EDTA tubes (i.., lavender stopper) 6 si Oxalate/fluoride tubes (i... gray stopper) 212. Skin Puncture Procedure (Rodak) 1. Blood gases 2. Slides, unless made from EDTA microcollection tube 3. EDTA microcollection tube 4, Other microcollection tubes with anticoagulants (i.., green or gray) 5. Serum microcollection tubes 213. Venipuncture procedure: (Steininger) 4. Patient interaction 2. Assemble supplies and equipment 3. Venipuncture 4. Specimen preparation 214. The venipuncture must site must be cleanse thoroughly with a commercial alcohol prep or a cotton ball or gauze pad soaked in 70% isopropanol. 215. Gauge numbers refers to the diameter, or bore of the needle. The higher the gauge, the smaller the diameter. Routinely, 19., 20- and 21- gauge needles are used. The needle length chosen is an individual preference. The most commonly used lengths are 1 and 1.8 inches. (Steininger) 216. The tourniquet should be securely wrapped around the unclothed arm about 7.5 to 10 cm (3 - 4 inches) above the selected site. (Steininger) 217. Wedge smear. Transfer a blood drop approximately 2 to 3 mm in diameter to the stationary slide about 0.25 inch from the end or frosted area, Position the spreader slide at a 25° angle to the stationary slide and bring it back into the drop of blood. (Steininger) 218. COUNTING METHODS Cross-sectional or crenellation | WECs are counted in consecutive fields as the blood film is moved from side to side Longitudinal method WBCS are counted in consecutive fields from the tail toward the head of the smear Battlement method Uses a pattem of consecutive fields beginning near the tail on a horizontal edge: count three consecutive horizontal edge fields, count two fields towards the center of the smear, count two fields horizontally, count two fields vertically to the edge. 219. When the white count is below 1 x 10°L, it may be difficult to find many white blood cells on the stained smear. In this situation, a differential may be performed counting 50 white cells. A notation on the report must then be made that only 50 white blood cells were counted, Although, alternatively, a buffy coat smeared may be prepared, 220. When the differential shows an abnormal distribution of cell types: a. Over 10% eosinophils b. Over 2% basohils, ©. Over 11% monocytes, or d. More lymphocytes than neutrophils except in children, a 200-cell differential may be performed. The results are then averaged (divided by 2) and a notation made on the report that 200 white blood cells were counted. 221. Excessively Blue Stain. Thick films, prolonged staining time, inadequate washing, or too high an alkalinity of stain or diluent tends to cause excessive basophilia. In such films, the erythrocytes appear blue or green, the nuclear chromatin is deep blue to black, and the granules of the neutrophils are deeply overstained and appear large and prominent. The granules of the eosinophils are blue or gray. Staining for a shorter time or using less stain and more diluent may correct, the problem. If these steps are ineffective, the buffer may be too alkaline, and a new one with a lower pH should be prepared.Page |29 222. Excessively Pink Stain. Insuficient staining, prolonged washing time, mounting the coverslips before they are dry, or too high an acidity of the stain or buffer may cause excessive acidophilia. In such films, the erythrocytes are bright red or orange, the nuclear chromatin is pale blue, and the granules of the eosinophils are sparkling brillant red. One of the causes of the increased acidity is exposure of the stain or buffer to acid fumes. The problem may be a low pH of the buffer, or it may be the methyl alcohol, which is prone to develop formic acid as a result of oxidation on standing. Henry) 223. Other Staining Problems. inadequately stained red cells, nuclei, or eosinophilic granules may be due to understaining or excessive washing, Prolonging the staining or reducing the washing may solve the problem (Henry) Precipitate on the fim may be due to unclean slides; drying during the period of staining; inadequate washing of the slide at the end of the staining period, especially failure to hold the slide horizontally during initial washing; inadequate filtration of the stain; or permitting dust to settle on the slide or smear. (Henry) 224. Human hematopoietic stem cells (HSCs) express CD34 but lack the major histocompatibility complex (MHC) class II antigen, HLA-DR. CD34 is a glycoprotein that is encoded on chromosome 1q and is expressed by hematopoietic stem cells, as well as early progenitor cells. (Henry) 225. Fat first develops in the shafts of long bones until the age of 18 years when the only active hematopoietic sites are in the pelvis, vertebrae, ribs, sternum, skull and proximal extremities of the long bones, (Steininger) 228. Myeloid metaplasia indicates that myeloid cells (granulocytes, erythrocytes and megakaryocytes) are produced in hematopoietic cites outside the marrow. Such production is also referred to as extramedullary hematopoiesis. These extramedullary sites are the same as those that formed blood cells in utero during the hepatic period of development including the liver, spleen and reticuloedothelial system. (Steininger) 227. Leukoerythroblastosis has several synonyms: leukoerythroblastic anemia, myelophthisic anemia and myelopathic anemia. Itis a condition of anemia caused by space occupying malignant tumors of the bone marrow. (Brown) 228. The Rapoport-Leubering pathway allows for the production of 2,3 diphosphoglycerate (2,3 DPG), which regulates the affinity of the hemoglbin molecule for oxygen. (Brown) 229. Diumal variation has been recognized in the neutrophil count, with highest levels in the afternoon and lowest levels in the morning at rest. (Henry) 230. BAND nucleus is elongated, curved or sausage-shaped with rounded ends and areas of dense clumping at each pole. (Steininger) 231. The maturation of the cells of the megakaryocytic system has bed divided into four stages. They are, from least to ‘most mature, the megakaryoblast, the promegakaryocytes, the megakaryocyte and the metamegakaryooyle. (Steiniger) 232. MORPHOLOGIC DIFFERENTIATION OF MEGAKARYOCYTIC CELL SERIES Maturation Stage Cytoplasmic Cytoplasmic Tags | Nuclear Features Thrombocytes Granules | Visible Megakaryoblast Absent Present Single nucleus, fine | No chromatin, nucleoli Promegakaryocyte Few Present Double nucleus No Megakaryocyte Numerous: Usually absent | Two or more nuclei No Metamegakaryocyte ‘Aggregated ‘Absent Four or more nuclel___| Yes 233. Platelets are difficult to count, They are small, disintegrate easily and are hard to distinguish from dirt, They readily adhere to each other (aggregation) and also become easily attached to any foreign body (adhesiveness). The use of EDTA as an anticoagulant helps to decrease platelet clumping, but the mean platelet volume will increase during the first hour in the tube. (Brown) 234. The phenomenon of “platelet satelitosis” may ocour when using EDTA anticoagulant. Platelets adhere around neutrophils, forming a ring or satellite effect. Using sodium citrate as an anticoagulant should correct this problem. Because of the dilution in the citrate tubes, itis necessary to multiply the obtained platelet count by 1.1. (Rodak) 235. Dilutional loss of platelets: Extensive blood transfusion often is accompanied by thrombocytopenia, the degree of which Is directly proportional to the number of units transfused. (Steiniger) 236. Platelet dysfunction and bleeding disorders will be present in the various paraproteinemias. In multiple myeloma and Waldenstrom’s macroglobulinemia, abnormalities of platelet aggregation and reduced platelet retention are thought to be due to the coating of the platelet membrane and vessel walls with the abnormal proteins. (Brown)Page 130 237. SMEAR: PLATELET ESTIMATES Platelet Est. of. Report Platelet Est, as: O— 49,000 Marked decreased 50,000 - 99, 000001 ‘Moderate decrease 100,000 - 149,000/uL. ‘Slight decrease 150,000 — 199,000/uL, Low normal "200,000 - 400,000/uL ‘Normal "401,000 ~ 599, 000%uL Sight increase {600,000 800,000/uL, Moderate increase ‘Above 800,000/iL ‘Marked increase 238. Platelets: The sol-gel zone lies directly beneath the platelet membrabe and is composed of microfilaments and microtubules. They provide a cytoskeleton to maintain platelet shape, and a contractile system. Microfilaments contain the proteins actin and myosin which, upon stimulation of the platelet, will interact to form actomyosin (thrombosthenin), a contractile protein, important in clot retraction, The microtubules are composed of the protein tubulin, which maintains the platelet's disc shape. (Brown) 239. Platelets: The surface connecting system (open canalicular system), as an invagination of the plasma membrane, acts as a canal for the release of the granule constituents and cytoplasm to the exterior of the platelet. (This system is also involved in platelet phagocytosis). (Brown) 240. The complete blood count (CBC) consists of the white blood cell count, red blood cell count, hemoglobin, hematocrit, and white blood cell differential. Also included are the red blood cell indices, which indicate the relative and absolute hemoglobin content and size of the average red blood cell. When performing the differential, the white blood cells are identified and categorized, all cells are examined for abnormalities and the platelets, are reviewed for number and morphologic features. (Brown) 241. A capillary Hct tube about 7 cm long with a uniform bore of about 1 mm is used. For blood collection directly from a skin puncture, heparinised capillary tubes are available. The microhematocrit tube is filed by capillary attraction from a freefiowing puncture wound or a well-mixed venous sample. The capillary tube should be filled to at least 5 cm. (Henry) 242. Microhematocrit method: Microhematocrit tube, approximately 75 mm long with an inner bore of approximately 1.2 mm, The microhematoorit tubes hold approximately 0.05 mL of whole blood. Seal one end of microhematocrit tube with the clay material by placing the dry end of the tube into the clay in a vertical position. The plug should be 4 to 6 mm long. Centrifuge for 5 minutes (ROF of 10,000 to 15,0009). (Brown) 243. The Wintrobe tube is 115 mm long with an internal bore of 3mm. For the sedimentation rate, the scale on the left side of the tube is used (0 is at the top). The scale on right (100 at the top) was historically used to read a macrohematocrt. (Brown) 244. The Westergren tube is a straight pipet 30 om long, 2.65 mm in internal diameter, and calibrated in milimeters from O- 200. (Henry) 245. Setting the rack of sedimentation rate tubes on top of the refrigerator could lead to the following: (Steininger) a. A falsely decreased ESR because of lower temperatures from air rushing out on opening the refrigerator or freezer b. A falsely increased ESR attributable to vibrations from opening and closing the refrigerator and freezer doors c._A falsely increased ESR because of heat released from the refrigerator motor. 246. ERYTHROCYTE SEDIMENTATION RATE ESR MARKEDLY INCREASED ESR MODERATELY INCREASED (100 MM OR MORE PER HOUR) Multiple myetoma ‘Acute and chronic infectious diseases ‘Waldenstrom's macroglobulinemia ‘Acute localized infections Malignant lymphoma Reactivation of a chronic infection Leukemia Rheumatic fever Acute and sever bacterial infection Rheumatoid artis Severe anemia Myocardial infarction Carcinoma Malignant tumors with necrosis Collagen diseases Hypertnyoidism Sarcoma Hypothyroidism Portal or biliary cirrhosis Lead and arsenic intoxication Ulcerative colts Nephrosis Severe renal disease Internal hemorrhage Acute hepatitis, Unruptured ectopic pregnancy afte third month Ruptured ectopic pregnancy Menstruation Normal pregnancy after third month Tuberculosis Ingestion of oral contraceptives Intravenous dextran Postcommissurotomy syndromeESR USUALLY NORMAL Page |31 ESR DECREASED Early acute appendicitis (within first 24 hours) Early unruptured ectopic pregnancy Malarial paroxysm Cirrhosis of iver Degenerative arthritis Infectious mononucleosis ‘Acute allergies Uncomplicated virus diseases Peptic ulcer Typhoid fever Undulant fever Rheumatic carditis with cardiac failure Pertussis ‘Spherooytosis Poikilocytosis, Sickle cell anemia Hemolytic jaundice ‘Severe iron deficiency Thalassemia 247. RULE OF THREE Basio Equations: 3X RBC = Hb 3 X Hb = Het + 3 (%) “rules apply only to red cells of normal size and color 248, Cyanmethemoglobin method: The color intensity of this mixture is measured in a spectrophotometer at a wavelength of 540 nm. The optical density of the solution is proportional to the concentration of hemoglobin. All forms of hemoglobin are measured with this method except sulfhemoglobin. (Brown) 249, DESCRIPTIVE TERMS: slight (less than 5%), moderate (5-15%), and marked (more than 15%) to denote variation in size, shape, or color content per average oil immersion field, If there is a particular size variation such as macrocytes, or a poikilocytes, such as schistocytes, the term occasional (less than 1%), few (1-5%), frequent (5-10%), and many (more than 10%) are used, SAMPLE CRITERIA FOR ERYTHROCYTE MORPHOLOGY EVALUATION Morphologie Characteristics WNL | a Macrocytes >9 um dia, 05 5:10 10-20 20-50 350 Microcytes <9 um dia 05 510 10-20 20-50 350 Hypochromia 02 310 10-50 50-75 375 Poikilocytosis (generalized variations in shape) 0-2 3-10 10-20 20-50 >50 Burr cells 02 310 10-20 20-50 350 ‘Acanthocytes <1 25 5-10 10-20 320 Schistocytes <1 25 5-10 10-20 320 Dacryocytes 02 25 5-10 10-20 320 Codocytes 02 240 0-20 20-50 >50 ‘Spherocytes 02 240 0-20 20-50 350 Ovalocytes 02 240 0-20 20-50 350 Stomatocytes 02 240 0-20 20-50 >50 Sickle cells ‘Abs Report as 1+ to indicate presence, do not quantitate Polychromatophilia Adult “1 25 5-10 10-20 >20 Newborn 16 TAS 15-20 20-50 >50 Basophilc sipping ot 18 5-10 10-20 320 Howell-Jolly bodies ‘Abs 12 35 5-10 310 ‘Siderocytes (Pappenheimer bodies) Abs 4-2 35 5-10 >10 Guidelines for semiquantitation are expressed in number of occurrences per oll immersion field (applies to fields oF ‘approximately 200 to 250 cells per 100x oil objective. WNL = Within normal limtsRED CELL MORPHOLOGY GRADING CHART MORPHOLOGY GRADE AS. Polychromatophilia T= 110 Sffield Helmet cells 2+ = 6 to 10/field Teardrop RBC 3+ = >t0/field Acanthocytes Schistocytes ‘Spherocytes Poikilocytosis, To Tomield Ovalocytes 1 to 20ifield Elliptocytes 20/field Burr cells Bizarre-shaped RBC Target cells StomatocytesPage [32 MORPHOLOGY GRADE AS Rouleaux 1+ = aggregates of 3 to 4 RBC 2+ = aggregates of 5 to 10 RBC ‘3+ = numerous aggregates with only few free RBC Sickie cells Grade as positive only Basophilic stippling Pappenheimer bodies Howell-Jolly bodies (GRADING SCALE FOR RED CELL HYPOCHROMIA GRADING POLYCHROMASIA GRADING MORPHOLOGY 4+ Area of central pallor is one- | Percentage of Red cells that are ANISOCYTOSIS/POIKILOCYTOSIS half of cell diameter Polychromatophilic Percentage of Red Cells that Slight 1% Differin Size or Shape fromNormal | 2+ Area of pallor is two-thirds of | >'8! RBs cell diameter 1+ 3% Slight 5-10% 3+ Area of pallor is three ad 5% ie 10.25% quarters 3+ 10% 2 25.50% 3+ 50-75% 4+ Thin rim of hemoglobin at >11% 4 575% 250. A Schistocyte with one or more horlike projections has been identified as a keratocyte. A keratocyte is the result of an 251 252. 253, 254, 256, 256, 257. 258, 259, 260, 261 262. 263, erythrocyte being caught on a fribrin strand, which could cut the cells in two, Keratocytes do not remain in circulation for more than few hours, as they are fragile. Serious bacterial infections are a major cause of death in patients with sickle cell anemia. (Brown) Infectious crises are a primary cause of death in sickle cell anemia. (Steininger) Broken Cells: Damaged or broken leukocytes constitute a small proportion of the nucleated cells in normal blood. Bare nuclei from ruptured cells vary from faitly well-preserved nuclei without cytoplasm to smudged nuclear material, sometimes with strands arranged in a coarse network, the so-called basket cells. They probably represent fragil cells, usually lymphocytes that have been broken in preparing the film. They are apt to be numerous when there is an atypical lymphocytosis, in chronic lymphocytic leukemia, and in acute leukemias. (Henry) After leukocyte alkaline phosphatase (LAP) staining, a Kaplow count is performed, a total of 100 mature neutrophils are scored from 0 (negative) to 4 (strongly positive), based upon the intensity of the staining reaction. (Hubbard) Increased LAP values are found during pregnancy (the last trimester), in infection accompanied by neutrophilia, polycythemia vera, people receiving corticosteroids, multiple myeloma, myelosclerosis, and obstructive jaundice. In Hodgkin's disease the results of this stain closely parallel the progression of the disease, being elevated in untreated cases. (Brown) Decreased LAP scores are found in chronic myelogenous leukemia, paroxysmal noctumal hemoglobinuria, sickle cell anemia, hereditary hypophosphatasemia, marked eosinophilia, sideropblastic anemia, and rarely, in normal people. (Brown) Normal alkaline phosphatase activity will be found in untreated haemolytic anemia, lymphosarcoma, viral hepatiti and secondary polycythemia. (Brown) Ham method for PNH: when the patient has received blood transfusions, less lysis occurs because of the presence of ‘normal transfused red blood cells. (Brown) Petechiae — purplish red pinpoint hemorrhagic spots in the skin caused by loss of capillary ability to withstand normal blood pressure and trauma Purpura — hemorhage of blood into small areas of skin, mucous membranes, and other tissues Ecchymosis - form of purpura in which blood escapes into large areas of skin and mucous membranes, but not into deep tissues Hematemesis — vomiting of blood Melena ~ stool containing dark red or black blood264. PROTEINS OF THE PLASMA COAGULATION SYSTEM Page |33 Surface-bound zymogens Vitamin K-dependent zymogens Cofactors/substrates Factor Xil Factor Vil HMWK Prekallkrein Factor IX Factor Vill Factor XI Factor X Factor V Factor Il Fibrinogen Protein C Protein S 265. FAMILIES OF COAGULATION PROTEINS THROMBIN-SENSITIVE PROTHROMBIN FAMILY CONTACT FAMILY COAGULATION PROTEINS I, V, Vill and XIII Il, VI, IX and X Xil, Xl, Not vitamin K dependent Vitamin K dependent rein, HMWK Consumed during coagulation Present in serum (except Il) Not vitamin K dependent Absent in serum Absent in adsorbed plasma Present in serum Present in adsorbed plasma Present in adsorbed plasma 266. Poor venipuncture technique causing hemolysis or tissue thromboplastin to mix with the blood shortens the clotting time. (Brown) 267. An abnormally shortened APTT may be caused by partial clotting of the blood as a result of a traumatic venipuncture, high levels of factor Vill, disseminated intravascular coagulation, or the presence of platelets in the plasma. (Brown) 268. Lee and White clotting time method Equipment: water bath 37°C; glass test tubes 13 x 100 mm; stopwatch and plastic syringe (10 mL) and 20-gauge needle. (Brown page 215) 269. Alll test tubes used for coagulation studies should have a noncontact surface. That is, the inside surface if the tubes should not be of a material; (such as glass or soda lime) that will react or activate the coagulation factors. Most vacutainer tubes for collecting coagulation studies have a siliconized surface. (Brown) 270. Brown Prothrombin Time: Test tubes, 13 x 100 mm 0.1 mL patient's plasma 0.2 mL (200 pL) thromboplastin-calcium reagent APTT: 13 x 100 mm tube 0. 2mL plasma 0.2 mL APTT reagent 0.2 mL CaCh 271. Steiniger Prothrombin Time: 0.1 mL plasma 0.2 mL (200 pL) PT reagent APTT: 12x75 mm tube 0.1 mL PPP 0.1 mL APTT reagent 0.1 mL Cach, 272. The closer the ISI is to 1, the more sensitive the reagent is; the higher the ISI, the less sensitive the reagent. 273. Increased levels of fibrin (ogen) split degradation products (FSP or FDP), as seen in DIC and lytic disorders, exert an anticoagulant effect. Laboratory procedures utilized to evaluate this process include latex FSP agglutination test, ‘measurements of fibrin monomers, platelet counts, fibrinogen levels, APTT and PT. (Steininger) 274. The D-dimer test is positive in DIC as soon as 4 hours after onset. Fibrinogen levels may decrease in 4 to 24 hours; platelets decrease up to 48 hours after onset. (Steiniger) 275. INSTRUMENTATION FOR TESTS OF HEMOSTASIS Visual detection of fibrin clot formation Tilt tube method Electromechanical detection of fibrin clot formation Fibrin strand formation is detected using a wire loop or hook; has been incorporated into a semi-automated mechanical instrument Instrument: FIBROMETER Photo-optical detection of fibrin clot formation Detection of fibrin clot formation depends on the increase in light scattering associated with the conversion of soluble fibrinogen molecules to the insoluble polymerized fibrin clot Semi-automated instruments: Electra 750 and 750A, Fibrintimer series, and FP 910 Coagulation Analyzer Automated instruments: Ortho Koagulab 16S and 40A, the Coag-A-Mate X2 and XC, and the MLA Electra 700 and 800Page [34 276. Electromechanical systems detect fully formed fibrin. These instruments (e.g., Fibrometer) require the operator to add all the reagents to a reaction cup, but temperature control and a timing device for clot detection are provided by the instruments. Timing is initiated either automatically through activation by the pipetor or manually by depressing the timer bar. (Steininger) 277. In a typical electrical impedance instrument by Coulter, aspirated blood is divided into two separate volumes for measurements. One volume is mixed with diluent and is delivered to the cell bath, where erythrocyte and platelet counts are performed. As a cell passes through the aperture, partially occluding it, the electrical impedance increases, producing @ voltage pulse, the size of which is proportional to the cell size. The number of pulses is directly related to the cell count. Particles measuring between 2 and 20 fL are counted as platelets, whereas those measuring greater than 36 fL are counted as erythrocytes. The other blood volume is mixed with diluents and a cytochemicallytic reagent that lyses only the red blood cells. A leukocyte count is performed as the remaining cells pass through an aperture. Particles greater than 35 fL are recorded as leukocytes. (Henry) 278. OHM'S LAW: voltage = current X resistance. The magnitude of the voltage pulses produced by cells is directly related to their size, a fact that has been used in subsequent clinical instruments for direct measurements of cell volume, 279. The MCV and RDW are derived from the RBC histogram, whereas the MPV and PDW are derived from the platelet histogram. The HCT, MCH, and MCHC are calculated from measured and derived values, (Steininger) 280. The analyzers computer deives the MCV and RDW from the red cell histogram. The MPV and platelet count are derived from the platelet histogram. The hematocrit, MCH and MCHC are computed. (Coulter STKS, Brown) 281, COULTER STKS: Sample results are routinely displayed on data management system (DMS) soreen: WBC scatterpiot, RBC and platelet histograms, numeric results for the CBC, RBC indices, ROW, platelet count, MPV, five cell differential and any abnormal flagging codes or messages. This report may also be printed by the graphic printer. (Brown) 282. The Coulter Counter S-Plus provides (in addition to the routine 10-parameter profiles) percentage and values of lymphocytes, mononuclear cells, and granulocytes, as well as leukocyte, erythrocyte, and platelet histograms. Leukocytes are sized electronically after they have had their cytoplasm and nucleus differentially shrunk by a special lysing agent and reagent system. The cells are then categorized into lymphocytes (small cells), mononuclear cells (medium cells), and granulocytes (large cells). (Steininger) 283. The use of propriety Iytic reagents, as used on the older Coulter S-Plus IV, STKR, and Sysmex E-5000 to control shrinkage and lysis of specific cell types, allow for separation and quanttation of white blood cells into three populations (lymphocytes, monocytes and granulocytes) for the “THREE-PART DIFFERENTIAL” on one size distribution histogram. (Rodak) 284. The use of multiple methods on a given instrument for the determination of at least two cell properties allows the separation of WBCs into a FIVE-PART DIFFERENTIAL (neutrophils, lymphocytes, monocytes, eosinophils, and basophils). (Rodak) 285. Technicon H Systems: A ratio of signals is used to deteremine the degree of lobularity of the nuclei. This information is used to indicate the degree of left shift present. (Brown) The left shift (unsegmented neutrophils) flag is derived from monitoring the value for the LI (lobularoty index) and analysis of the separation of mononuclear and polymorphonuclear populations in the baso/lobulanty cytogram. (Brown) 286. Potential errors from cell counting instrument can be categorized in two types: INSTRUMENTAL ERRORS 1. Aperture plugs are probably the most common problem in cell counting (positive error) 2. Extraneous electrical pulses from improperly grounded or shielded equipment may be picked up by the instrument electrode ( positive error) 3. Improper setting of aperture current or threshold (either a positive or negative error) 4. Bubbles in the sample caused by too vigorous mixing (positive error) 5. Excessive lysing of RBCs (negalive error) ERRORS CAUSED BY THE NATURE OF THE SPECIMEN 1. Giant platelets may be counted as RBCs or WBCs 2. Fragments of leukocyte cytoplasm, such as may be present during leukemia therapy, may be counted as platelets or RBCs Increased number of schistocytes may make accurate erythrocyte and platelet count impossible Agglutination of erythrocytes, leukocytes or platelets will cause false negative results for each of the respective cell counts Agglutinated red cells or platelets may also cause false positive leukocyte counts Platelet satelitism will result in falsely low platelet counts Some abnormal RBCs tend to resist lysis, which may result in high WBC counts. Examples sickle cells, extremely hypochromic cells and target cells. The problem can be solved by delaying 2 to 3 minutes between the addition of the lysing reagent and counting. (Steininger)MICROBIOLOGY AND PARASITOLOGY Page 138 ‘The chain of infection requires a continuous link between three elements: a source, a method of transmission, and a ‘The 6 components in the infectious disease process (also known as the chain of infection) are: source of infection (the pathogen), reservoir, portal of exit, mode of transmission, portal of entry, and susceptible host. (Burton) 287, susceptible host.(Stevens) 288, 289._ PLATING MEDIA FOR ROUTINE BACTERIOLOGY MEDIUM PRIMARY PURPOSE Bile esculin agar (BEA) Bile esculin azide agar with vancomycin Differential isolation and presumplive identification of group D streptococci and enterococci ‘Selective and differential for cultivation of vancomycin- resistant enterococci from clinical and surveillance specimens Blood agar Cultivation of infectious microorganisms, determination of hemolytic reactions Bordet-Gengou agar Isolation of Bordetella pertussis Buffered charcoal-yeast extract (BCYE) agar Enrichment for Legionella spp. Buffered charcoal-yeast extract (BCYE) agar with ‘Campylobacter thioglycollate broth ‘Cefoperazone, vancomycin, amphotericin (CVA) medium Enrichment and selection for Legionella spp. ‘Seleclive for Campylobacter spp. Selective holding medium for recovery of Campylobacter spp. ‘Selective medium for isolation of Campylobacter spp. Cefsulodin-irgasan-novobiocin (CIN) agar Selective for Yersinia spp.; may be useful for isolation of ‘Aeromonas pp. Chocolate agar Cultivation of Haemophilus spp. and pathogenic Neisseria spp. ‘Columbia colistin-nalidixic acid (CNA) agar Selective isolation of gram-positive cocci Cystine-tellurite blood agar Isolation of C. diphtheriae Eosin methylene blue (EMB) agar (Levine) isolation and differentiation of lactose-fermenting and non- lactose fermenting enteric bacilli ‘Gram-negative broth (GN) Selective (enrichment) medium for enteric pathogens Hektoen enteric (HE) agar Differential, selective medium for isolation and differentiation of Salmonella and Shigella spp.from other gram-negative enteric bacilli MacConkey agar MacConkey sorbitol agar Isolation and differentiation of lactose-fermenting and non- lactose fermenting enteric bacili For the selection and differentiation of E. coll O187:H7 in stool specimens Mannitol salt agar Selective isolation of staphylococci New York City (NVC) agar Selective for N.gonorrhoeae Phenylethyl alcohol (PEA) agar Seleclive isolation of gram-positive cocci and anaerobic gram-negative bacili Regan Lowe Enrichment and selective medium for isolation of B. pertussis ‘Salmonella-Shigella (SS) agar Selective for Salmonella and Shigella spp. ‘Schaedler agar Selenite broth Skirrow agar ‘Streptococcal selective agar (SSA) Tetrathionate broth Thayer-Martin agar Thioglycollate broth Nonselective medium for the recovery of anaerobes and aerobes Enrichment of isolation of Saimonella spp. ‘Selective for Campylobacter spp. Selective for S. pyogenes and S. agalactiae Selective for Salmonella and Shigella spp. Selective for N. gonorrhoeae and N, meningitidis ‘Supports growth of anaerobes, aerobes, microaerophilic and fastidious microorganisms ‘Thiosulfate citrate-bile salt sucrose (TCBS) agar Selective and differential for Vibrio spp. ‘Todd-Hewitt broth supplemented with antibiotics Trypticase soy broth Selective and enrichment for S. agalactiae in female genital specimens Enrichment broth used for subculturing various bacteria from primary agar plates Xylose lysine desoxycholate (XLD) agar Isolation and differentiation of Salmonella and Shigella spp. from other gram-negative enteric baci ‘Source: Bailey and Scolfe Diagnostic WerobileayPage 136 290. COLONIAL APPEARANCE AND CHARACTERISTICS (Bailey and Scott's) ‘Organism [Medium | ‘Appearance Citrobacter spp. Mac Late LF, therefore NLF after 24 hours; LF after 48 hours; colonies are light pink after 48 hours HE | Colorless | XLD_| Red, yellow or colorless colonies with or whithout black centers (HS) Edwardsiella spp. Mac | NLF HE | Colorless XLD __| Red, yellow or colorless colonies with or without black centers (H:S) Enterobacter spp. Mac | LF, may be mucoid HE Yellow XLD__| Yellow Escherichia coli Mac LF; flat, dry, pink colonies with surrounding darker pink area of precipitated bile salts* HE Yellow XLD__| Yellow Hafnia alvei Mac | NLF HE | Coloriess. XLD__| Red or yellow Klebsiella spp. Mac | LF, mucoid HE | Yellow XLD__| Yellow ‘Morganella spp. Mac | NLF HE | Colortes XLD__| Red or colorless Proteus spp. Mac | NLF; may swarm depending on the amount of agar in the medium, characteristic foul smell HE | Colorless | XLD__| Yellow or colortess, with or without black centers Providencia spp. Mac | NLF HE | Colorless XLD__| Yellow or colorless ‘Salmonella spp. Mac | NLF HE | Green XLD__| Red with black center ‘Serratia spp. Mac | Late LF; S. marcescens may be red pigmented, especially i plate is lef at 25°C HE | Coloriess XLD__| Yellow or colorless ‘Shigella spp. Mac | NLF; S.sonnei produces flat colonies with jagged ends HE | Green XLD__| Colorless Yersinia spp. Mac | NLF; may be colorless to peach HE | Salmon XLD__| Yellow or colorless HE: Hektoen enteric agar, ‘Mac: MacConkey agar LF: lactose fermenter, pink colony NLF: non-lactose fermenter; XLD: Xylose-tysine-desoxycholate agar “Most Enterobacteriaceae are indistinguishable on blood agar Pink colonies on MacConkey agar with sorbitol are sorbitol fermenters; colorless colonies are nonsorbitol fermenters 291. Xylose-Lysine-Desoxycholate (XLD) Agar. XLD agar is selective and differential media for Shigella spp. and ‘Salmonella spp. The salt, sodium desoxycholate, inhibits many gram-negative bacilli that are not enteric pathogens and inhibits gram-positive organisms, A phenol red indicator in the medium detects increased acidity from carbohydrate (lactose, xylose and sucrose) fermentation. Enteric pathogens, such as Shigella spp., do not ferment these carbohydrates, so their colonies remain coloriess (i. the same approximate pink to red color of the uninoculated medium). Colonies of Salmonella spp. are also colorless on XLD, because of the decarboxylation of lysine, which results in a pH increase that causes the pH indicator to turn red. These colonies often exhibit a black center that results from ‘Salmonella spp. producing HS. Several of the nonpathogens ferment one or more of the sugars and produce yellow colonies. (Bailey and Scot's) 292. IMVIC REACTIONS Escherichia E.coli + +Page |37 Klebsiella T w Vv c K pneumoniae - = + + K oxytoca + Vv + + K ozaenae : + L 5 Vv Enterobacter T wt Vv c E_ aerogenes = Si + + E. cloacae 7 = + + E_ agglomerans Vv Vv Vv Vv 293. Grade A milk is obtained only from healthy (tuberculin negative) cows. Its bacterial content is always below 75,000 bacteria per ml when raw and should not exceed 15,000 bacteria per ml once pasteurized. (Albas) 204. Certified milk is produced under close medical supervision. It contains 10,000 or less bacteria per mil. (Albas) 295. lodine tincture: iodine in alcohol (Bailey and Scott’s) 296. BLOOD SPECIMENS: Two or three separate samples from different venipunctures paced at least 1 hour apart are recommended. The maximal number of culture sets that may be ordered in a 24-hour period is determined by each microbiology laboratory; most facilities set a maximum of three or four sets of blood cultures per 24 hours, unless ‘a unique situation exists, (Delost) 297. The Schaeffer-Fulton stain is a technique designed to demonstrate endospores by staining any present endospores green. 298. Staphylococcus - white or golden colony, grape-like clusters 299. Slide coagulase detects bound coagulase. Tube coagulase detects free coagulase. 300. S. saprophyticus is a cause of urinary tract infection in young females. 301. Beta-hemolytic streptococci produce two hemolysisn: ‘Streptolysin S - oxygen stable, non-antigenic Streptolysion O - oxygen labile, antigenic 302. Group B beta haemolytic streptococci is a major pathogen in newborn.tt is CAMP positive and hippurate hrdrolysis positive BACTERIAL IDENTIFICATION AND STRATEGIES TEST POSITIVE RESULT | NEGATIVE RESULT POSITIVE NEGATIVE CONTROL CONTROL, ‘Acetamide Utilization | Deamination of the No color change Pseudomonas ‘Stenotrophomonas acetamide resulting in a aeruginosa ‘mattophilia | blue color. | | ‘Acetate Utilization | Medium becomes No growth or growth | Escherichia colt ‘Shigella flexneri alkaline (blue) because | with no indicator ofthe growth ofthe | change to blue organism, Bacitracin Test ‘Any zone ofinhibition | No Zone of inhibition | Streptococcus ‘Streptococcus agalactiae around the disk pyogenes Bile Esculin Agar | Blackening of the agar _| No Dlackening ofthe | Enferococcus ‘Streptococcus mitis slant medium, faecalis Bile Solubility Test | Colony disintegrates; | Intact colonies Streptococcus Enterococcus faecalis an imprint of the lysed pneumoniae colony may remain within the zone | | Buiyrate disk Development of a blue | No color change ‘Moraxella catarrhalis | Neisseria gonorrhoeae color during 5-minute | incubation period. _| | CAMP Enhanced hemolysis is_| No enhancement of | Streptococcus ‘Streptococcus pyogenes indicated by an hemolysis agalactiae arrowhead shaped zone of beta hemolysis, at the juncture of the two organisms.Page |38 TEST POSITIVE RESULT | NEGATIVE RESULT POSITIVE NEGATIVE CONTROL CONTROL, Catalase Test Copious bubbles No or few bubbles | Staphylococcus ‘Streptococcus pyogenes produced produced, aureus Cetrimide Growin No growth Pseudomonas Escherichia col aeruginosa Citrate Utilization Growth inthe medium | Absence of growth | Klebsiella Escherichia col with or without a pneumoniae change in the color of the indicator. The color change of the indicator is due to acid or alkali production by the test ‘organism as it grows on the medium. Growth usually results in the bromthymol blue indicator turning from | green to blue. | | Coaguiase Test Cot of any size No clot ‘Staphylococcus ‘Staphylococcus epidermidis aureus Decarboxylase Tests | Alkaline (purple) color | No color change or __| Lysine: Klebsiella | Lysine: Enterobacter (Moeller's Method) | change compared with | acid (yellow) color in| pneumoniae cloacae the control tube. test and control tube. | Ornithine: Omithine: Klebsiella Enterobacter pneumoniae cloacae Arginine: Klebsiella Arginin pneumoniae Enterobacter Base: Klebsiella cloacae pneumoniae Base: — DNA Hydrolysis When DNAs Tihere is no ‘Staphylococcus ‘Staphylococcus epidermidis hydrolyzed, methyl degradation of DNA, | aureus greens released and | the medium remains combines with highly | green. polymerized DNA at a pH of 7.5, turning the medium colorless around the test organism, Esculin Hydrolysis | Blackened medium No blackening and | Klebsiella ‘Shigella flexneri which would also show | no loss of pneumoniae aloss of fluorescence | fluorescence under under the Wood's lamp. | wood’s lamp, or slight blackening with no loss of fluorescence under ‘Wood's lamp. Fermentation Media: | Indicator changes to | Growth but no Dextro: Peptone medium with | pink with or without gas | change in color, Positive, with gas: | Pseudomonas aeruginosa ‘Andrade’s indicator ( | formation in Durham | Medium remains Escherichia coli for enteric and tube clear to straw- Positive, no gas: coryneforms) colored. Shigella flexneri Fermentation Media: | Indicator change to | Growth, but no Sorbitol: Heart infusion broth | yellow change in color, Streptococcus Streptococcus mitis with bromeresol Medium remains ‘mutans purple indicator (for purple. streptococci and enterococci) Flagella Stain (Wet Mount Technique) EXPECTED RESULTS: Observe the slide and note the following = Presence or absence of flagella = Number of flagella per cell Location of flagella per cell aeruginosa Kiebsiella pneumoniae Gelatin Hydrolysis Partial or total ‘Complete liquefaction of the solidification of tube inoculated tube (the | at 4°. control must be completely solidified at 4°C within 14 days) Proteus vulgaris Enterobacter aerogenesge 139 Test POSITIVE RESULT | NEGATIVERESULT | POSITIVE NEGATIVE CONTROL CONTROL, Growth at 42°C ‘Good growth No growth Pseudomonas Pseudomonas fluorescens aeruginosa Fippurate Test Deep purple color Colorless or slightly | Streptococcus ‘Streptococcus pyogenes yellow pink color | agalactiae Indole Production Pink-to-wine colored ring after addition of No color change afer the addition of Kovac’s metho: Klebsiella pneumoniae Kovac’s method: Escherichia coli Kovac's reagent, the appropriate Ehrlich's method: | Ehrlich's method: CDC reagent Elizabetnkingia ‘group £0-2 | | |_meningoseptica LAP Test Development of a red | No color change or | Enterococcus Leuconostoc sp. color within 1 minute | development of a faecalis, after adding slight yellow color cinnamaldehyde reagent Litmus Wik EXPECTED RESULTS Appearance of Indicator (Litmus Dye) COLOR pH CHANGE TO RECORD Pink, mauve ‘Aad ‘Reid (A) Blue Alkaline ‘Alkaline (K) Purple (dentical to ‘No change No change uninoculated control) ‘White dependent of pH, result of Decolorized eduction of indicator ‘Appearance of Milk ‘CONSISTENCY OF MILK ‘OCCURS WHEN pH is RECORD Coagulation oF clot ‘Acid or alkaline Clot Dissolution of clot with clear, | Acid Digestion grayish, watery fluid and a shrunken, insoluble pink clot Dissolution of clot with clear, | Alkaline Peptonization grayish, watery fluid and a shrunken, insoluble blue clot QUALITY CONTROL: Alkaline: Aicaligenes faecalis, ‘Acid: Enterococcus faecium Peptonization: Burkholderia cepacia Microdase Test Development of blue to | No color change purple-blue color Micrococcus luteus | Staphylococcus aureus Motility Testing Hanging drop In Brownian movement, the organisms may appear quite active, but they remain in the same relative position to other organisms or debris in the field In true motility, the organisms change in position with respect to each other, often darting across the field Escherichia col ‘Klebsiella pneumonulae Motility Testing Semisolid agar Motile organisms will spread out into the Nonmotile organisms remain at the site of Escherichia col ‘Klebsiella pneumonulae deep medium from the site of | inoculation | inoculation | | MRS Broth Gas production No gas production | Leuconostoc spp. | Pediococcus sp. indicated by a bubble in the Durham tube MUG Test Electric blue Lack of fluorescence | Escherichia col Pseudomonas aeruginosa fluorescence Nitrate Reduction | EXPECTED RESULTS NOs", no gas: ‘Acinetobacter Sp. The nitrate reduction test is read for the Escherichia coli presence or absence of three metabolic NOs", gas: products: gas, nitrate (NO:), and nitrite (NO;). | Pseudomonas aeruginosa Nite Reduction No color change to red | The broth becomes | Alcaligenes faecalis _| Alcaligenes plechaudii 2 minutes after the addition of reagents and gas production observed in the Durham tube. red after the addition of the reagents. No {gas production is observed.Page [40 TEST POSITIVE RESULT | NEGATIVE RESULT POSITIVE NEGATIVE CONTROL CONTROL, ‘ONPG. Yellow Clear Escherichia col ‘Salmonella typhimurium ‘Optochin Zone of inhibition is 14 | No zone of inhibition | Streptococcus ‘Streptococcus mitis mm or greater in pneumonia diameter, with 6mm. disk, ‘Oxidase Test Development of a dark | Absence of color Neisseria Escherichia col (Kovac's method) | purple color within 10 gonorrhoeae seconds Phenylalanine Green color develops | Slant remains Proteus vulgaris | Escherichia cof Deaminase on slant after ferric, original color after chloride is added. the addition of ferric chloride, PYR Test Bright red color within 5 | No color change or __| Enferococcus ‘Streptococcus mitis minutes an orange color faecalis Pyruvate Broth Indicator changes from | No color change; Enterococcus Enterococous faecium green to yellow. yellow-green faecalis, indicates a weak | | reaction, | Salt Tolerance Visible turbidity in broth | No turbidity and no | Enferococcus ‘Streptococcus mitis with or without color | color change faecalis, change from purple to yellow ‘Spot Indole Test Development of a blue | No color Escherichia col Enterobacter cloacae color within 20seconds | development or slightly pink color Urea Hydrolysis ‘Change in color of slant | No color change Proteus vulgaris | Escherichia cof Christensen’s from light orange to. | (agar slant and butt Method magenta Femain light orange) X and V Factor Test | Growth around XV Growth over the A. influenzae will | A. aphrophilus will grow disks only shows entire surface of the | show halo growth | over the entire surface of requirement for both | agar indicates no | around XV disk; the | the plate. Neither X nor V factors. Growth around | requirement for rest of the agar nor XV factors are V disk, no growth either X or V factor. | surface will show no | necessary for growth. around the X disk, and growth light growth around the H. parainfluenzae XV disk shows a V will show a halo factor requirement. growth around the XV and V disks. 303. Acetamide utilization test: This test is used to determine the ability of an organism to use acetamide as the sole source of carbon. Bacteria that can grow on this medium deaminate acetamide to release ammonia. The production of ‘ammonia results in a pH-driven color change of the medium from green to royal blue. a. Inoculate acetamide slant with a needle using growth from an 18- to 24- hour culture, Do not inoculate from a broth culture, because the growth will be too heavy. b. Incubate at 35C for up to 7 days ©. Positive: P. aeruginosa d. Negative: Stenotrophomonas maltophilia (Bailey and Scot's) 304. Acetate utilization test: This test is used to determine if an organism can use acetate as the sole source of carbon. If so, breakdown of sodium acetate causes the pH of the medium to shift toward the alkaline range, turning the indicator from green to blue. a. With a straight inoculating needle, inoculate acetate slant lightly from an 18- to 24- hour culture. Do not inoculate from a broth culture, because the growth will be too heavy. . Incubate at 38C for up to 7 days ©. Positive: E. coli d. Negative: S. flexneri (Bailey and Scott's) 305. Malonate utilization test uses sodium malonate in phosphate broth with bromthymol blue indicator. Some organisms can utilize sodium malonate as the sole source of carbon, Positive: blue Negative: green or yellowPage |41 306. MUG test: Escherichia coli produces the enzyme beta-D-glucuronidase which hydrolyzes beta-D-glucopyranosid-uronic derivatives to aglycons and D-glucuronic acid. the substrate 4-methylumbellferyl-beta-D-glucuronide is impregnated in the disk ang is hydrolyzed by the enzyme to yield the 4-methylumbelifery| moiety, which fluoresces blue under long wavelength ultraviolet light. ‘Wet the disk with one drop of water. Using a wooden applicator stick, rub a portion of a colony from an 18- to 24-hour-old pure culture on the disk Incubate at 35 °C in a closed container for up to 2 hours. ‘Observe disk using a 366-mm ultraviolet light. Expected results Positive: electric blue fluorescence Negative: lack of fluorescence f. Quality control Positive: E.coli Negative: P.aeruginosa 307. Phenylalanine deaminase is an enzyme that removes the amino group from an amino acid Phenylalanine agar ‘Overnight culture + 10% ferric chloride Positive: green slant and fluid Negative: no color change 308. Oxidase test: a. 1% tetramethyl-p-phenylenediamine dihydrochloride, prepared fresh daily b. Remove colony with platinum loop or wooden applicator 1. Iron in the nichrome loop can give a false positive reaction 2. Rub colony on a paper strip and add a drop of reagent; or 3. Rub the colony on a piece of paper containing the reagent; or 4, Puta drop of reagent on the colony cc. Positive - turns DARK PURPLE in 10 seconds. 309. Decarboxylase: Enzyme that removes the carboxyl group (COOH) from an amino acid Uses decarboxylase basal medium with indicator (bromeresol purple) + sugar (glucose) + amino acid (Iysine, ornithine, arginine) + control tube with no amino acid Moller's medium better Positive: purple to yellow to purple 310. M. catarrhalis may be carried in the oropharynx of healthy children and adults. It is an encapsulated organism, and extending from its outer membrane are pili that serve as adhesins. The most common infections caused by this organism are bronchitis, otitis, sinusitis, and pneumonia. Neatly allisolates of M. catarrhalis produce B-lactamase, which can be detected using nitrocefin, Although they should be assumed to be resistant to penicillin because of this, these isolates generally remain susceptible to cephalosporins, trmethoprim-sulfamethoxazole, and f-lactamase inhibitor combinations. (Henry) 311. Horse or rabbit blood agars are commonly used for detecting hemolysis by hemolysin-producing strains of Haemophilus that will not grow on 5% sheep blood. (Bailey and Scott's) 312. Mycobacterium tuberculosis is highly resistant to drying, when protected from sunlight they remain in putrifying sputum for weeks and in dried sputum for 6-8 months. Droplets of dried sputum in the air may be infectious for 8-10 days. Organisms from culture are killed within two hours when exposed to direct sunlight; but in sputum, they require 20-30 hours exposure before they are killed. They are generally more resistant to chemical disinfection than other vegetative organisms; specifically difficult to disinfect in sputum which requires 24 hours exposure in 5% phenol. But they are easily killed by moist heat; boiling for 10 minutes, pasteurization or steam under pressure (autoclave). (Training Manual on Direct Sputum Microscopy, National Tuberculosis, Reference Laboratory, RITM, DOH) 313. Lepromin test is a skin test for leprosy using a sterile extract from lepromatous nodules. Two types of reaction: early or Femandez reaction (24 to 48 hours) and the late or Mitsuda reaction (3 to 4 weeks). 314. Lepra cells are macrophages containing acid-fast bacill 315. Strains of C. albicans produce germ tubes from their yeast cells when placed in a liquid nutrient environment and incubated at 35C for 3 hours (similar to the in vivo state). (Bailey and Scott's) 316. Candida albicans is the only yeast that produces a germ tube when incubated with Sterile SErUM for 1 to 3 hours al 35° to 37°C. A germ tube is a hyphal extension of the yeast cell wth no constriction atthe point of origin, (Delost) 317. The antigens of H. capsulatum are similar to those of B.dermatitidis, occasionally a patient with Histoplasmosis will demonstrate positive reactions with serologic tests for 8. dermatitis (Bailey and Scotts) 318. All adenovirus types except those of avian origin have a group reactive antigen, the hexon. (PER)Page [42 319. Rotavirus is the most important cause of acute viral gastroenteritis worldwide (50% of hospitalized cases in children, during winter months). (PER) 320. Calcium alginate should be avoided for collection of samples for viral culture, because it could inactivate herpes simplex virus (HSV); cotton may be toxic to Neisseria gonorrhoeae; and wooden shafts should be avoided, because the wood may be toxic to Chlamydia trachomatis. Swabs are not optimal for detection of anaerobes, mycobacteria, or fungi, and they should not be used when these organisms are suspected. An actual tissue sample or fluid aspirate is always superior to a swab specimen for the recovery of pathogenic organisms. (Henry) 321. UNHOLY THREE: Hookworm, large roundworm and whipworm. (PER) 322. Trematodes (flukes) require 2 intermediate hosts except for the Schistosomes (only 1) 1°. — snail 2° LH. ~ fish, crab, plant/vegetation, snail Fish: Heterophyes heterophyes, Clonorchis sinensis, Opistorchis felineus Crab: Paragonimus westermani Plantivegetation: Fasciola hepatica, Fasciola gigantica, Fasciolopsis buski Snail: Echinostoma ilocanum 323. Paragonimus westermani: The fisit intermediate hosts are snails of the genera_Hua, Semisulcospira, Syncera, and Thiara in the Far East, Pomatiopsis in North America, and possibly Pomacea in South America. The second intermediate hosts are the freshwater crabs of the genera Eriocheir, Potamon, Sesarma, and Parathelpusa in the Far East, and Pseudothe/pusa in South America, and crayfishes of the genus Astacus in the Far East and Cambarus in North America and probably elsewhere. (Neva and Brown) 324. Pulmonary paragonimiasis has been described among patient diagnosed to have PTB and not responding to treatment and among patients suspected to have PTB in Sorsogon. transmission is mainly attributed to the preparation Of local delicacies (e.g. kinagang in Sorsogon) using crab juice, eating of undercooked roasted crabs by children and eating of raw crabs by adult males with local alcoholic drinks. (Belizario, de Leon) 325. Schistosome eggs are relatively easy to identify and have terminal (Schistosoma haematobium), lateral (S. mansoni), or small lateral (S japonicum) spines. (Bailey and Scott's) 326. Spirometra species and their spargana have been reported in animals in the Philippines. Spargaum is widespread in tadpoles and frogs and has been found in a bird. Adult Diphyllobothrium latum has been reported locally from a boy ‘who died of anemia, Since D. fatum is only found in temperate countries of the Northern Hemisphere, this identification is doubtful. tis possible that the specied in question is Spirometra erinacei or S. mansonoides. (Belizario, de Leon) 327. The adult Taenia seems to be irritated by alcohol and passage of proglottids results after a drinking bout. (Belizario, de Leon) 328. Taenia: Specific diagnosis rests on identifying the characteristic proglottids, eggs or scolex. The first specimen usually brought in by patients are the gravid progiottids, either single or in chains. Gravid proglottids are pressed or flattened in between two glass slides and are examined against the light. This will allow one to have rough count of the lateral branches from the main uterus. Injection of India ink through the genital pore will help one make an accurate count of the lateral branches of the uterus (15 to 20 for T. saginata and 7 to 15 for T.solium), T. saginata Tsolium Gravid proglottid | Lateral branches (dichotomous or tree-like) Lateral branches (dendritic or fingeriike) 329. A third Taenia sp. was reported to ocur among the aborigines of Taiwan. By the adult morpholohu, the so-called "Taiwan" Taenia resembles T. saginata, but by the appearance of the cysticercus, itis close to T.solium. In the current taxonomy, this parasite is known as Taenia asiatica. 330. Hymenolepis nana eggs are spherical or subspherical measuring 30 to 47 jum in diameter. The oncosphere has a thin outer membrane and a thick inner membrane with conspicuous bipolar thickenings, from which arise 4 to 8 hair-like polar filaments embedded in the inner membrane. 331. Hymenolepis diminuta eggs are circular, about 60 to 80 jim in diamter and are bile-stained. The oncosphere is enclosed in an inner membrane, which has bipolar thickenings but lacks the bipolar filaments. The hooklets usually have a fan-like arrangement. 332. Beyond 1 hour, the stool must be refrigerated. (PER) 333. Trophozoites can no longer be seen in iodine-stained preparation since they are destroyed by iodine. (PER) 334. Important risk factors for Giardiasis include: poor hygiene, poor sanitation, overcrowding, immunodeficiency, bacterial and fungal overgrowth in the small intestine, and homosexual practices, Giardiasis has been shown to be related to the “GAY BOWEL SYNDROME”. (Belizario, de Leon)Page 143 335. Rapid Diagnostic Tests (RDTs) for malaria detect either histidine rich protein 2 (HRP-2) which is only produced by P. falciparum or the parasite lactate dehydrogenase (pLDH) produced by all four Plasmodium species. All asexual stages of the parasite secrete both antigens in the blood. (Belizario, de Leon) 336. There are several parasites that may be recovered from the sputum, These include: migrating larva of A. lumbricoides, S. stercoralis, and the hookworms; Paragonimus ova; E. granulosus hooklets from pulmonary hydatid cysts; protozoa such as: E. histolytica trophozoites from pulmonary abscesses, C. parvum oocysts (although very rare), non-pathogenic E. gingivalis and T. tenax. (Belizario, de Leon) 337. Kato Thick Smear: About 50 to 60 mg of stool (approximately the size of two mongo beans) is placed over a glass slide and covered with cut cellophane paper soaked in a mixture of glycerine and malachite green solution. Glycerine is a clearing solution and malachite green is used to give color to the cellophane in order to give a pale green background to the eggs and to minimize the brightness of the microscopic field. Without malachite green, green cellophane may also be used. The preparation is best examined within 10 to 20 minutes. The technique is simple and economical, therefore useful in mass stool examinations. It is very good in detecting eggs with thick shell (Ascaris, Trichuris) but not eggs with thin shells (nookworm). 338. Examination of blood: Several species of helminthic parasites (filarial) and protozoan parasites (Plasmodi Trypanosomes and Babesia) are in the blood at some stage of their development. a. Finger-prick blood sample must be free-flowing to prevent dilution of blood with tissue fluid, which decreases number of parasites, ‘+ Wetffresh preparation - microfilaria and trypomastigotes are large and motile in fresh blood preparations ‘+ Stained smears - thick films (2 to 3 small drops of blood: 2 cm in diameter), thin smears © Stains that are generally used for blood parasites include: Giemsa, Wright's and Delafield hematoxylin stain © Capillary tube technique - microfllariae and trypanosomes can be readily visualized at the buffy coat area b. Venous blood may be concentrated in order to detect microfilariae 339. Phthirus pubis (genital louse; pubic/crab louse) has a crab-like body. It is found on axillary hair, eyelashes, eyebrows, and other parts of the body where the hair is courser and more widely spaced, as compared to the hair of the head 340. A study conducted in UP-Manila College of Public Health in 1981 recovered the following parasite pathogens from Periplaneta Americana (cockroach): Ascaris, Trichuris, and parasites under family Thelastomatidae and Superfamilies Spiruroidea and Tylenchoidea. Other pathogens include Proteus, Escherichia, Salmonella and Citrobacter. (Belizario, de Leon) IMMUNOLOGY-SEROLOGY AND BLOOD BANKING 341. NOBEL PRIZE WINNERS IN IMMUNOLOGY (Stevens) YEAR ‘SCIENTIST RESEARCH 1901 | Emil von Behring Serum antitoxins 1905 __| Robert Koch Cellular immunity in TB 7908 | Elie Metchnikof, Phagocytosis Paul Ehrlich Immunity 1913 _| Charles Richet ‘Anaphylaxis 1919 | Jules Bordet Complement 1930 _| Karl Landsteiner Human blood group antigens 1960 _| Macfarlane Burnet, Peter Medawar Discovery of immunologic tolerance 1972 _| Gerald Edelman, Rodney Porter Structure of antibodies 1977 | Rosalyn Yalow Radioimmunoassay 1980__| George Snell, Jean Dausset, Baruj Benaceraf Major histocompatibiity complex 1984 | Niels Jere immunoregulation Georges Koehler, Cesar Milstein Monoclonal antibody 7987__| Susumu Tonegawa ‘Antibody diversity 1991 | Edward Donnall Thomas, Joseph Murray ‘Transplantation 1996__| Peter Doherty, Rolf Zinkernage! Cytotoxic T cell recognition of virally infected cals 72008 __| Francoise Barré-Sinoussi, Luc Montagnier Human immunodeficiency virus 342. HISTORICAL PERSPECTIVES (Stanley) 1798 | Edward Jenner, an English countryside physician demonstrated that protection from cowpox could be generated by the transfer of postural material from a cowpox lesion instead of the more hazardous smallpox lesion 1880 | Louis Pasteur demonstrated that injection of Killed microbes provided protection upon subsequent exposure to Ive counterpart 71888 | Elie Metchnikoff demonstrated that certain blood cells ingest foreign material 1894 | Jules Bordet discovered complement 1897 | Robert Kaus discovered precipitins 1901 | Emil von Behring had the distinction of being awarded as the first immunology-related Nobel Prize for his works on serum therapyPage [4a 1984 | Discovery of the T cell receptor gene 1987 | Susumu Tonegawa was awarded the Nobel Prize for his 1978 discovery of the genetic principles underlying the generation of antibodies with different specificities 343. The first written records of immunological experimentation date back to the 1500s, when the Chinese developed a practice of inhaling powder made from smallpox scabs in order to produce protection against this dreaded disease. This practice of deliberately exposing an individual to material from smallpox lesions was known as variolation, The theory was that if a healthy individual was exposed as a child or young adult, the effects of the disease would be minimized. (Stevens) 344, Further refinements did not occur until the late 1700s, when an English country doctor by the name of Edward Jenner discovered a remarkable relationship between exposure to cowpox and immunity to smallpox. After observing the fact that milkmaids who were exposed to cowpox had apparent immunity to smallpox, he deliberately injected individuals ‘with material from a cowpox lesion and then exposed them to smallpox. He thus proved that immunity to cowpox, a very mild disease, provided protection against smallpox. This procedure of injecting cellular material became known as vaccination, from vacca, the Latin word for “cow.” (Stevens) 345. The pre-Columbian theory states that syphilis was present in Europe prior to the voyage of Columbus, but was not recognized as such, was confused with other diseases (e.g., leprosy) or was present in milder form than seen today. Columbian theory states that syphilis was endemic in Haiti and was subsequently contracted and carried to Europe by Columbus crew. (Bryant) 346. Elie Metchnikoff, a Russian scientist, observed that foreign objects introduced into transparent starfish larvae became surrounded by motile cells that attempted to destroy these invaders. He called this process phagocytosis, meaning cells that eat cells. Stevens) 347. In 1492, blood was taken from three young men and given to the sticken Pope Innocent VII in the hope of curing him; unfortunately, all four died, Although the outcome of this event was unsatisfactory, itis the first time a blood transfusion was recorded in history. (Harmening) 348. The most famous study of haptens was conducted by Karl Landsteiner, a German scientist who was known for his discovery of the ABO blood groups. In his book The Specificity of Serological Reactions, published in 1917, he detailed the results of an exhaustive study of haptens that has contributed greatly to our knowledge of antigen-antibody reactions. (Stevens) 349. Porter's work was based on the use of the proteolytic enzyme papain, which was used to cleave IgG into three pieces of about equal size, each having a sedimentation coefficient of 3.5 S and representing a molecular weight of approximately 45,000 to 50,000 d. (Stevens) 350. Alfred Nisonoff used pepsin to obtain additional evidence for the structure of immunoglobulins. This proteolytic enzyme was found to cleave IgG at the carboxy-terminal side of the interchain disulfide bonds, yielding one single fragment with ‘a molecular weight of 100,000 d and all the antigen-binding ability, known as F(ab)2. An additional fragment called Fc ‘was similar to Fc except that it disintegrated into several smaller pieces. (Stevens) 351. The segment of H chain located between the CH1 and CH2 regions is known as the hinge region. It has a high content of proline and hydrophobic residues; the high proline content allows for flexi (Stevens) 352. HOST DEFENSES IN INNATE IMMUNITY Cells Antigen presenting cells, basophils, eosinophils, mast cells, natural killer cells, phagooyles Humoral Factors ‘Complement proteins, lactoferrin, lysozyme, pepsin, stomach acidity ‘Anatomical Barriers | Cilia, mucus, skin Resident flora Mainly non-pathogenic bacteria HOST DEFENSES IN ADAPTIVE IMMUNITY Cells B cells, plasma cells, T cytotoxic cells, T helper cells Humoral Factors ‘Antibodies, cytokines 353. Phagocytes can interact with microorganisms directly via primitive pattern recognition receptors (PPRP) that recognize a wide array of molecules present on the surface of microorganisms. Phagocytes have also been shown to interact indirectly with microorganisms via opsonins that have been deposited on the microbial cell surface. 354. Macrophages have specific names according to their particular tissue location. Some are immobile, while others progress through the tissues by means of amoeboid action. Macrophages in the lung are alveolar macrophages; in the liver, Kupffer cells; in the brain, microglial cells; and in connective tissue, histiocytes. (Stevens) 355. Dendritic cells are so named because they are covered with long membranous extensions that make them resemblenerve cell dendrites. Their main function is to phagocytose antigen and present it to helper T lymphocytes. While their actual developmental lineage is not known, they are believed to be descendents of the myeloid line. They are classified according to their tissue location, in a similar manner to macrophages. Langerhans cells are found on skin and mucous membranes; interstitial dendritic cells populate the major organs such as the heart, lungs, liver, kidney,Page [45 and the gastrointestinal tract; and interdigitating dendritic cells are present in the T lymphocyte areas of secondary lymphoid tissue and the thymus. (Stevens) 356. Eosinophils are capable of phagocytosis but are much less efficient than neutrophils because of the smaller numbers present and their lack of digestive enzymes. Their most important role is neutralizing basophil and mast cell products and killing certain parasites. (Stevens) 367. Diapedesis: The process by which cells are capable of moving from the circulating blood to the tissues by squeezing through the wall of a blood vessel. (Stevens) 358. _ Characteristics of Acute-Phase Reactants PROTEIN RESPONSE TIME (hr) | NORMAL. INCREASE | FUNCTION CONCENTRATION (mg/dL) C-reactive protein | 6-10 0.5 1000x. Opsonization, complement activation Serum amyioid A [24 30 1000x Removal of cholesterol Alphat-antitrypsin | 24 200-400 2-5x, Protease inhibitor Fibrinogen 24 10-400 2-5x [ Clot formation Haptogiobin 24 40-200 ‘2-10x [Binds hemoglobin Geruloplasmin | 48-72 20-40 2x [Binds copper and oxidizes iron Gomplement C3_| 48-72 60-140 2x [Opsonization, isis Mannose-binding |? 0.15-1.0 ? Complement activation protein 369. Proinflammatory cytokines found in synovial fluid that contribute to inflammation are interleukin- 1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-1 (IL-1), interleukin-18 (IL-18), and tumor necrosis factor-alpha (TNF-c). (Stevens) 360. Interleukin-8 is an activating and chemotactic factor for neutrophils and to, a lesser extent, for eosinophils, and lymphocytes. Therefore, IL-8 and related cytokines may serve as the principal secondary mediators of inflammation, (Bryant) 361. Interleukin-3 supports the growth of pluripotential stem cells of the hematopoietic system. (Bryant). 362. Latex agglutination test for CRP. CRP latex reagent, which contains a 1 percent suspension of polystyrene latex particles coated with anti-human CRP produced in goats or rabbits. (Stevens) 363. The classical pathway of complement activation, described around the year 1900, was the first to be studied, and for this reason is called “classical.” (Henry) 364. DEFICIENCIES OF COMPLEMENT COMPONENTS DEFICIENT COMPONENT T ‘ASSOCIATED DISEASE C1(q, Fors) | Lupusiike syndrome: recurrent infections 2 | Lupusiike syndriome: recurrent infections, atherosclerosis 3 [ Severe recurrent infections; glomerulonephritis cA | Lupusiike syndrome coc8 ‘Neisseria infections co [No known disease association CHINA | Hereditary angioedema DAF [Paroxysmal noctumal hemoglobinuria MIRE [Paroxysmal noctumal hemoglobinuria Factor H or factor! | Recurrent pyogenic infections MBL Pneumococcal diseases, sepsis, Neisseria infections Properdin ‘Neisseria infections MASP-2 Pneumococcal diseases 365. The deficiency that occurs most often is that of C2, which is found in 1 in 20,000 individuals. Recent evidence indicates that atherosclerosis may be related to a C2 deficiency, Additionally, C2-deficient individuals may be more prone to recurrent streptococcal and staphylococcal infections. The factor B locus is nearby, and often C2-deficient Persons are reported to have decreases in factor B. Other types of complement deficiencies are less common. (Stevens) 366. The most serious deficiency, however, is that of C3, because it is the key mediator in all pathways. Individuals with a C3 deficiency are prone to developing severe, recurrent life-threatening infections with encapsulated bacteria such as Streptococcus pneumoniae and may also be subject to immune complex diseases. Such complexes can be lodged in the kidney and result in glomerulonephrtis. (Stevens) 367. Some studies indicate that a DAF deficiency is associated with a lack of CD59 (MIRL), and both are implicated in Paroxysmal Nocturnal Hemoglobinuria (PNH). CDSS has the same glycophospholipid anchor found in DAF, so the gene deficiency affects both molecules. CD59 prevents insertion of C9 into the cell membrane by binding to the C5b678Page |46 complex, thus inhibiting formation of transmembrane channels. Therefore, the presence of both DAF and CD59 is important in protecting red blood cells against bystander lysis. (Stevens) 368. The strongest link found to date between certain HLA molecules and specific diseases Is between HLA-B27 and ankylosing spondylitis. Individuals who possess HLA-B27 have a 90 times greater chance of developing the disease than the normal population. (Stevens) 369. Allogenic (allogeneic): Genetically different individuals of the same species. 370. The cellular approach to tissue matching uses the mixed leukocyte reaction (MLR). The MLR is an in vitro test sed to mimic in vivo condition of transplantation. (Stanley) 371. Selective IgA deficiency is the most common of the immunodeficiency disorders, (Stanley) 372. Bladder cancer tumor markers for the management of patient with bladder cancer have been actively investigated, Assays approved for clinical use include the following: = Matritech nuclear matrix protein (NMP-22) + Bard's BTA test (Bladder tumor-associated antigen (Turgeon) 373. Coagglutination is the name given to systems using bacteria as the inert particles to which antibody is attached, Staphylococcus aureus is most frequently used, because it has a protein on its outer surface, called protein A, which naturally adsorbs the fragment crystallizable (FC) portion of antibody molecules. The active sites face outward and are capable of reacting with specific antigen. These particles exhibit greater stability than latex particles and are more refractory to changes in ionic strength. However, because bacteria are not colored, reactions are often difficult to read. Such testing is highly specific, but it may not be as sensitive for detecting small quantities of antigen, as is latex agglutination, Coagglutination reagents have been used in identification of streptococci, Neisseria meningitidis, Neisseria gonorrhoeae, Vibrio cholera 0139, and Haemophilus influenzae. (Stevens) 374. The INHIBITION IMMUNOFLUORESCENT ASSAY is a blocking test in which antigen is first exposed to unlabeled antibody, then to labeled antibody, and is finally washed and examined. If the unlabeled and labeled antibodies are both homologous to the antigen, there should be no fluorescence. This result confirms the specificity of the FA technique. Antibody in an unknown serum can also be detected and identified by the inhibition test. (Turgeon) 375. Wester biot is used as a confirmatory test to detect antibodies to human immunodeficiency virus. A mixture of HIV antigens is placed on a gel and electrophoresed to separate the individual components. The components are then transferred to nitrocellulose paper by means of blotting or laying the nitrocellulose over the gel so that the electrophoresis pattem is preserved. Patient serum is applied to the nitrocellulose and allowed to react. The strip is then washed and stained to detect precipitin bands. It is simpler to visualize the reaction on the nitrocellulose, and in this manner, antibodies to several antigens can be detected. (Stevens) 376. Analysis of DNA fragments using probes is typically carried out using a technique known as a Southern hybridization assay, or Southern blot, named after its discoverer, E. M. Southern. After DNA fragments are separated out by gel electrophoresis, the fragments are denatured using alkali and then transferred, or “blotted,” onto a nitrocellulose or ylon membrane for the hybridization reaction to take place. (Stevens) 377. Treponema pallidum immobilization (TPI) test: If §0 percent or more of the treponemes are immobilized, the test is regarded as positive. if fewer than 20 percent are immobilized, the test is negative. The range of 20 to 60 percent represents an area of ‘doubtful'result. (Bryant) 378. When using RF latex tests, a titer of 80 or greater us generally considered a positive reaction, a titer of 20 to 40 is considered a weakly positive reaction, and, if there is no agglutination at 1:20, the specimen should be considered negative for RF, even if subsequent dilution shows agglutination. (Bryant p279). 379, fal agglutination test: The results are scored from 0 to 4+ where 0 represents no agglutination, When agglutination is present, the results are graded as +1, +2, +3, or +4 which represent 25, 50, 75 and 100 percent agglutination, respectively, A single rapid slide test is not considered positive unless a value of at least +2 is assigned in a sample teaken in the first 2-3 weeks of infection. (Stanley) 380. Quantitative tests are performed by RT-PCR, realtime PCR, or branched DNA amplification (bDNA). They are used to monitor the amount of HCV RNA, or “viral load,” carried by patients before, during, and after antiviral therapy in chronically infected individuals. The ultimate goal of such therapy is to achieve a sustained virological response (SVR), in which the patient continuously tests negative for HCV RNA 6 months after therapy is completed. The initial viral load level has also been used as a prognostic tool, since those with a low initial viral load are most likely to achieve an SVR. (Stevens)Page |47 381. Davidsohn designed a classic differential test to distinguish between heterophil sheep cell agglutinins in human serum due to Forssman antigen, serum sickness, and infectious mononucleosis. (Bryant) ADSORPTION PATTERNS IN DAVIDSOHN DIFFERENTIAL TEST TYPE OF HETEROPHILE ANTIBODY ‘ADSORBED BY ADSORBED BY GUINEA PIG KIDNEY CELLS BEEF ERYTHROCYTES Forssman ‘Yes No ‘Serum sickness Yes Yes Infectious mononucleosis No. Yes 382. The OptiMal Assay is an immunochromatographic (dipstick) assay that uses monoclonal antibodies to detect parasitic lactate dehydrogenase (pLDH) present in a finger prick sample. This test is based on the fact that the enzyme lactate dehydrogenase is produced by viable malaria parasite present in infected red blood cells and in circulation. Distinction between species (P. falciparum versus P. vivax) is possible because different isoforms of lactate dehydrogenase are present in different species, Using this assy, pLDH can be detected when there are 100-200 parasites/ul blood, 383, MalaQuick Standby Malaria Test detects the P. falciparum histidine-rich protein-2 (HRP-2) antigen. 384. Three E. histolytica structural proteins have been identified as antigenic and capable of eliciting antibody responses, These are (1) the 170 kD subunit of the lectin protein (adhesion), (2) the serine-rich Entamoeba histolytica protein (SREHP) and (3) a 2kD cysteine-rich protein, 385. Serum or plasma may be used for pretransfusion testing. Most blood bank technologists prefer serum because plasma ‘may cause small fibrin clots to forms, and these may be difficult to distinguish from true agglutination. Also, plasma may inactivate complement so that some antibodies may not be detected. About 10 ml of blood is usually sufficient for all testing procedures if there are no known serologic problems. (Harmening). 386. The minor crossmatch test has been completely eliminated in most blood banks because donor samples are screened beforehand for the more common antibodies. The presence of low-incidence antibody in the donor's plasma probably would not cause a transfusion reaction because it would be diluted in the recipient's plasma (Harmening 4” ed.) 387. Both donor and recipient samples must be stored for a minimum of 7 days following transfusion. The samples should be stoppered, carefully labelled, and refrigerated at 1 to 6C. They should be adequate in volume so they can be re- evaluated if the patient experiences any adverse reaction to the transfusion. (Harmening) 388. ISBT TERMINOLOGY FOR RED BLOOD CELL SURFACE ANTIGENS IN BLOOD GROUP SYSTEMS ISBT SYSTEM ‘SYSTEM ‘CHROMOSOMAL NUMBER NUMBER 001 ‘ABO 9 004 Rh. 1 389. ABO DISCREPANCIES GROUP | DISCREPANCIES: WEAKLY REACTING OR MISSING ANTIBODIES 1. Newborns Elderly patients Patients with leukemia demonstrating hypogammaglobulinemia Patients with lymphomas demonstrating hypogammaglobulinemia Patients using immunosuppressive drugs that yield hypogammaglobulinema Patients with congenital agammaglobulinemia Patients with immunodeficiency diseases Patients with bone marrow transplantations (develop hypogammaglobulinemia GROUP Il DISCREPANCIES: WEAKLY REACTING OR MISSING ANTIGENS 1. Subgroups of A and/or B 2. Leukemias (weakened A or B antigens) 3. Hodgkin's disease 4, Excess amounts of blood group-specific soluble substances (BGSS) present in the plasma in association with certain diseases such as carcinoma of the stomach and pancreas ‘Acquired B phenomenon Antibodies to low incidence antigens. GROUP Ill DISCREPANCIES: PROTEIN OR PLASMA ABNORMALITIES RESULTING TO ROULEAUX FROMATION 1. Elevated levels of globulin from certain disease states: MM, Waldenstrom’s macroglobulinemia, other plasma cell dyscracias, advanced Hodgkin's lymphoma 2. Elevated levels of fibrinogen 3. Plasma expanders such as dextran and polyvinylpyrrolidone (PVP) 4. Wharton's jellyPage |48 GROUP IV DISCREPANCIES: MISCELLANEOUS 1 2 3 4 5 Polyagalutination Cold reactive antibodies Unexpected ABO isoagglutinins Antibodies other than anti-A and anti-8 may react to form antigen-antibody complexes that may then adsorb into patient's red cells RBCs with the cis “AB phenotype” 390. Washing cord cells six to eight times with saline should alleviate spontaneous rouleaux due to Wharton's jelly, a viscous mucopolysaccharide material present on cord blood cells, and should result in an accurate RBC grouping. (Harmening p126) 391. ACQUIRED B ANTIGEN is associated with conditions such as carcinoma of the colon or rectum, intestinal obstruction, massive infection of the lower gastrointestinal tract, and septicemia caused by Proteus vulgaris. t can also result from increased permeability of the intestinal wall and subsequent absorption of bacterial polysaccharide (Escherichia coli (as) on the erythrocyte 392. Most naturally occurring ABO antibodies are of IgM isotype, although IgA and IgG antibodies with ABO specificity are also present. (Henry) 393. Para-Bombay: Red cells of these individuals express weak forms of A and b, which are primarily detected by adsorption and elution studies. If a person is genetically A or B, the respective enzymes can be detected, but no H enzyme is detectable. The small amount of H substance on the RBC is completely used by the A andlor B transferase present. This results in small quantities of A and/or B antigen being present on the RBC with no detectable H antigen. These individuals are designated as A, B,, or AB}. (Harmening 4” Ed.) 394, FISHER-RACE PHENOTYPES OF THE RH SYSTEM: FREQUENCIES IN THE UNITED STATES Gene nou combination White Black American Asian bce 42 ic aes Fo a o 37 26 a 3 bee “4 44 o. 21 dCe 2 Bj 3 = cE 1 ° 6 © DCB 0 ° 6 1 acer ° 0 iS a Widmann,“p 130. with permission. “Frequency less than om type has been found. 7 . One ee 395. Direct Antiglobulin Test: Although any red cells may be tested, EDTA-anticoagulated blood samples are preferred to prevent in-vitro fixation of complement. If red cells from a clotted blood sample have a positive DAT due to Complement, the results should be confirmed on cells from a freshly collected or EDTA-anticoagulated specimen if those results are to be used for diagnostic purposes. (AABB) 396. Some manufacturers market AHG colored with green dye for use in automated cell washers so that it will be immediately obvious if no reagent has been added, (ABB) 397. U phenotype U for universal ‘Antigen is located on GPB very close to the REC membrane High incidence antigen found on RBCs of individuals except 1 percent of American blacks (and 1 to 35 percent of ‘African blacks) who lack GPB 398. Anti-U Rare but can be formed in S- s- individuals Can also HDN and transfusion reactions Enhanced with enzyme treatment 399, Stimulation of Red Cell Autoantibody Production ~ Drug Independent: About 20% of hypertensive patients who receive methyldopa (Aldomet) for more than 3-6 months will eventually develop a positive DAT, but only 0.8% will develop a hemolytic anemia The DAT gradually becomes negative after stopping methyldopa treatment, although it may take ‘months to more than 2 years, Serologically, autoantibodies eluted from RBCs, or free in the serum, are indistinguishable from autoantibodies found in patients with WAIHA. The antibodies are usually of the IgG class, with both x and A light chains, and many show Rh specificity. One possible mechanism is that the drug binds to the cell membrane and sublly alters the structure, forming neoantigens that stimulate autoantibody formation. About 10% of patients with Parkinson's disease receiving L-dopa, a closely related drug, also develop RBC autoantibodies, but these rarely result in overt hemolysis. (Henry)Page [49 400. Autoantibodies with Kidd specificity are rare; but they have been associated with autoimmune haemolytic anemia Some examples are drug-related: one was found on a patient taking alpha-methyidopa (Aldomet); another was chlorpropamide-cependent. (Harmening p184) 401. High-incidence antigen Jk3 is present on any RBC positive for Jk” and Jk”. (Harmening) 402. People with the null phenotype lack Jk’, Jk®, and the common antigen Jk3. Although very rare, the Jk (a-b-) phenotype is most abundant among Polynesians, and it has also been identified in Filipinos, Indonesians, Chinese, and Japanese. (Harmening) 403. Alloanti-Jk3 is an IgG antiglobulin-reactive antibody that looks like an inseparable anti-JK°Jk”. The individual making the antibody will ype Jk (a-b-), 404. Lutheran antigen have been recognized since 1945, when the first example of anti-Lu® was discovered in the serum of a patient with lupus erythematosus diffuses, following the transfusion of a unit of blood carrying the corresponding low- incidence antigen. The new antibody was named Lutheran, a misinterpretation of the donor's name Luteran. (Harmening) 405. Data from human and old and new world monkey RBCs and their susceptibility to invasion by P. vivax and P. knowlesi indicate that Fy® is important for invasion for P. vivax, (Harmening) 408. Di? antigen has served as a useful tool in anthropolagic studies of Mongolian ancestry (Harmening) 407. Di and Di? antigens are located on the anion exchange molecule (AE-1), which is an integral transport protein involved in the anion exchange of bicarbonate for chloride in the red blood cell membrane. Mutations in AE-1 can result in hereditary spherocytosis, congenital acanthocytosis, and Southeast Asian ovalocytosis. (Harmening) 408. It was postulated that the CH/RG antigens were associated with the human leukocyte antigen (HLA) system; alleles for RG and CH have been located on two closely linked genes known as C4A and C4B on chromosome 6. Antigen products have been demonstrated on the Céd fragments of the C4A (Rodgers) and C4B (Chido) glycoproteins of the C4 ‘complement component. (Harmening) 409. Formery, the anti-Ch/Rg antibodies were collectively grouped as HIGH-TITER, LOW-AVIDITY (HTLA), along with other antibodies sharing common serologic properties. (Harmening) 410. The Cromer system antibodies are rarely observed, with the majority of examples presenting in black individuals. (Harmening) 411. Private antigen: an antigenic characteristic of the red blood cell membrane that is unique to an individual or a related family of individuals and, therefore, is not commonly found on all cells (usually less than 1% of the population). (Harmening) 412. Public antigen: an antigen characteristic of the red blood cell membrane found commonly among individuals, usually ‘more than 98% of the population. (Harmening) 413, Skin lesions: The skin at the site of venipuncture must be free of lesions, Both arms must be examined for signs of repeated parenteral entry, especially multiple needle puncture marks and/or sclerotic veins as seen with drug use. Such evidence is reason for indefinite exclusion of a prospective donor. Mild skin disorders or the rash of poison ivy should not be cause for deferral unless unusually extensive and/or present in the antecubital area. Individuals with boils, purulent wounds, or severe skin infections anywhere on the body should be deferred, as should anyone with purplish- red or hemorrhagic nodules or indurated plaques suggestive of Kaposi's sarcoma, (ABB) 414, If a potential donor has received a live attenuated or bacterial vaccine such as measles (rubeola), mumps, polio, typhoid, or yellow fever, there is a 2-week deferral; if the donor has received a live attenuated vaccine for German measles (rubella) or chickenpox, there is a 4-week deferral. There is however, no deferral for toxoids or killed or synthetic viral, bacterial or ricketsial vaccines ifthe donor is symptom-free and afebrile. 415. Donors indefinitely deferred: Sex with anyone since 197 who was bom in or lived in Cameroon, Central African Republic, Chad, Congo, Equatorial Guinea, Gabon, Niger or Nigeria, The basis for this deferral is the risk of exposure via sexual contact and the widespread use of nonsterile needles in these countries. (Harmening) 416. The FDA recommends travellers to areas of the world affected by SARS be deferred from donating blood for 14 days after their retum to US. The regions affected include the People's Republic of China, Hanoi, Vietnam, Singapore, and Toronto, Canada. Individuals who have had an acute case of SARS will be deferred from donating until 28 days after becoming asymptomatic and after any prescribed treatment is complete. 417. Any donor whose symptoms are consistent with the diagnosis of AIDS or ARC must be indefinitely deferred a. Persistent night sweats b. Fever of greater than 100.5F for more than 10 days ©. Unexplained weight loss of 10 Ib or more in less than 2 months d._Lymphadenopathy (swollen lymph nodes in the neck, armpits or groin)Page |50 e. Discolored areas of the skin (bluish purple areas under the skin typical of Kaposi's sarcoma) or white spots in the mouth or mucous membranes (typical or opportunistic thrush infection), or both f. Persistent diarrhea that lasts for several days or weeks and retums frequently g. Persistent cough or shortness of breath 1h. Malaise (fatigue, loss of energy) 418. Whole blood collection and phlebotomy: With the needle at 45° angle to the skin, make a quick clean puncture. Once in the skin, reduce the angle of the needle to about 10° to 20°, orient the ine of the vein, and make a second push through the vein wall; thread the needle up the vein about one-half inch to aid in securing the needle. (Harmening 4” Ed.) 419. Blood and blood products are considered drugs because of their use in treating diseases. As with drugs, adverse effects may occur, necessitating careful consideration of therapy. The transfusion of blood cells is also transplantation, in that the cells must survive and function after transfusion to a therapeutic effect. (Harmening) 420. Chronically transfusion-dependent patients should be exposed to as few units of RBCs as possible. One promising strategy is hypertransfusion using units rich in neocytes (young RBCs) to reduce the frequency of transfusion. 421. Apheresis or hemapheresis - to separate or remove, to take away (Harmening/AABB) 422. Procedures for performing apheresis vary according to the particular component of blood to be harvested and the equipment used. The amount of time for a particular procedure can range from 45 minutes to 120 minutes. (Harmening) 423. All apheresis procedures use anticoagulants to prevent blood from clotting as it enters the separation mechanism. The ‘most common anticoagulant is acid citrate dextrose, although heparin is occasionally used. (Harmening) 424. In 1943, Loutit and Mollison of England introduced the formula for the preservative acid-citrate-dextrose (ACD), (Harmening) 425. In 1987, Gibson introduced an improved preservative solution, citrate-phosphate-dextrose (CPD), which was less acidic and eventually replaced ACD as the standard preservative used for blood storage. (Harmening). 426. Reissue of Blood. If a unit of blood is retumed to the blood bank, it cannot be reissued unless certain requirements have been met: a. The container closure has not been disturbed. b. The blood has not been allowed to warm above 10 °C or cool below 1°C during storage or transportation. If the temperature of offsite storage (e.g., operating room refrigerator) cannot be documented, the blood should not be ‘way from the blood bank for more than 30 minutes, The records indicate that the blood has been reissued The blood is inspected prior to reissue. @. A sealed segment of integral donor tubing has remained attached to the container if the blood has left the premises ofthe issuing facilty f. Ifthe blood has remained on the premises of the issuing facility, a removed sample may be reindentified by confirming that the originally attached label and identification corresponds with the identification on the container. Turgeon) 427. Plasmapheresis: In manual procedures for donors weighing 50 to 80 kg (110-176 Ib), no more than 500 mL of whole blood should be removed at one time, or 1000 mL during the session or within a 48-hour period. The limits for donors ‘who weigh more than 80 kg are 600 mL and 1200 mL, respectively. For automated procedures, the allowable volume has been determined for each instrument by the FDA. (ABB) 428. The anticoagulant most often used for neonate transfusions is CPDA-1 429. Leukocyte concentrates, primarily neurophils may be of value to patient with severe neutropenia who have life- threathening systemic infection uncontrolled by antibiotics. Standards specify thet granulocytes be stored at 20 to 24°C. without agitation for 24 hours. 430. FEBRILE NONHEMOLYTIC TRANSFUSION REACTION: The AABB Technical Manual defines FNHTR as a 1°C temperature rise associated with transfusion and having no medical explanation other than blood component transfusion, (Harmening) 431. Bacterial multiplication is more likely in blood components stored at room temperature than in refrigerated components. Organisms that muttiply in refrigerated blood components are often psychrophilic gram-negative organisms (such as Yersinia enterocolitica, Serratia liquifaciens, and Pseudomonas fluorescens). Gram-positive organisms are more often seen in platelets stored at 20 to 24 C. For RBCs, the CDC estimates a symptomatic contamination rate of approximately 1 case per million units, primarily with Y. enterocolitica, followed by S. liquifaciens. In New Zealand, the incidence of symptomatic Yersinia contamination of RBC units has been reported to be as high as one in 65,000 units, with a fatality rate of one in 104,000. (AABB) 432. Deleterious conditions should be suspected if: 1) segments appear much lighter in color than what is in the bag (for AS- RBCs), 2) the red cell mass looks purple, 3) a zone of hemolysis is observed just above the cell mass, 4) clots are visible, 5) blood or plasma is observed in the ports or at sealing sites in the tubing, or (6) the plasma or supematant fuidPage |51 is murky, purple, brown, or red. A green hue from light-induced changes in bilirubin pigments need not cause the unit to be rejected. Mild lipemia, characterized by a milky appearance, does not render a donation unsuitable provided that all infectious disease testing can be performed. Grossly lipemic specimens are unsuitable. (ABB). 433. Units of blood that cannot be released for transfusion should be returned to the provider or discarded as biohazardous material. The nature of the problem disqualifying the unit should be investigated and the results reported to the blood supplier. Disposal procedures must conform to the local public health code for biohazardous waste. Autoclaving or incineration is recommended. (AABB) 434. Gel Technology: a. Advantages: standardization (major), decreased sample volume needed for testing, enhanced sensitivity and specificity, improved productivity, standardization, and ability to meet regulatory requirements when compared with traditional tube testing b. Disadvantage: need to purchase special incubators and centrifuges to accommodate microtubes used for testing, ‘Specific pipettes AGGLUTINATION REACTION IN THE GEL TEST ‘4+ | Solid band of agglutinated red cells at the top of the gel column. Usually no red cells are visible in the bottom of the microtube. 3+ | Predominant amount of agglutinated red cells towards the top of the gel column with a few agglulinates staggered below the thicker band, The majority of agglutinates are observed in the top half of the gel column, 2+ | Red cell agglutinates dispersed throughout the gel column with few agglutinates at the bottom of the microtubes. Agglutinates should be distributed through the upper and lower halves of the gel. 1+ | Red cell agglutinates predominantly observed in the lower half of the gel column with red cells also in the bottom. ‘These reactions may be weak, with a few agglutinates remaining in the gel area just above the red cell pellet in the bottom of the microtube. Negative | Red cells forming a well-delineated pellet in the bottom of the microtube. The gel above the red cell pellet is clear and free of agglutinates, Mixed- | Layer of red cell agglutinates at the top of the gel column accompanied by a pellet of unagglutinated cells in the field _| bottom of the microtube. 435, HIV: Symptoms may occur 6 to 12 weeks of infection and persist for a few days to 2 weeks. (Harmening) 436. HIV: The window period is that time after infection but before antibody or antigen is detectable by currently available testing procedures. Only a very small number of donors donate in the window period, about 1 in 4 millon. It is possible for a donation to be infectious but to test negative for HIV-1/HIV-2 antibodies when the donor is in the window period Antibodies are detectable at about 22 days after infection. (Harmening) 437. HIV: NAT testing has reduced the window period to about 12 days, according to FDA. (Harmening) 438. HIV: Criteria for determining a positive test result have been published by the Association of State and Territorial Public Health Laboratory Directors and CDC, the Consortium for Retrovirus Serology Standardization, the American Red Cross, and the FDA. Although some controversy exists as to what banding pattem constitutes a positive result, most laboratories follow the criteria of the Association of State and Territorial Public Health Laboratory Directors and CDC. According to these criteria, a result should be reported as positive if at least two of the following three bands are present: p24, gp41, and gp120/gp160. HISTOPATHOLOGY AND MEDICAL TECHNOLOGY LAWS 439. A computer system includes three major components: hardware, soft ware and people. Hardware components are the physical pieces of the equipment. Software is a set of instructions written in special computer language that tells the computer how to operate and manipulate the data, The people interface with the hardware to enter the data that are manipulated by the software. (Harmening) 440. Hardware components include a central system unit, sometimes referred to as the “box’, and a number of different peripheral devices that send or receive information through the system unit. Peripheral devices include display terminals, keyboards, bar code readers, scanners and wands, pointing devices such as mice, printers and modems. (Harmening) 441. Minimally, every computer system has two kinds of software - operating system software and application software. ‘Some systems may also use interface software, which allows the system to communicate with other computer systems. 442. Picric acid is explosive when dry or when cobined with metal and metallic salts. Pioric acid solutions, including yellow rinse fluids or processing solvents should not be disposed by pouring down the drain since they may form explosive picrates with metal pipes. 443. Sodium azide is very toxic and may be fatal when swallowed or absorbed through the skin, or when mixed with acid, It can explode when placed in contact with metals, and should not be discarded down the drain, 444. Silver salts are relatively safe when used as fresh solution, but can be explosive when solution becomes oldPage |S2 445. Aside from being expensive, dioxane is also extremely dangerous, and this is its main disadvantage. Its vapor produces a cumulative and toxic action in man. It should not be recycled as the risk of creating explosive peroxides increases greatly. 446._FIXATIVES ACCORDING TO COMPOSITION (Gregorios) ‘Aldehydes Formaldehyde 10% Formolsaline 10% Neutral buffered formalin or phosphate-butfered formalin (pH 7) Formol-corrosive (formol sublimate) Alcoholic Formalin (Gendre's) fixative Glutaraldehyde Metalllic Fixatives ‘Merccuric chloride Zenker’s fluid Zenker-formol (Helly's fluid) Heidenhain's Susa solution B-5 fixative, Metalllic Fixatives Chromate fixatives Chromic acid Potassium dichromate Regaud's fluid Onth's fluid Lead fixatives Picric Acid Fixatives Bouin's solution Brasi's alcoholic picroforrol fixative Alcohol Fixatives Methyl alcohol 100% Isopropyl alcohol 95% Ethyl alcohol 70-100% Camoy's fluid Newcomer's fluid ‘Osmium Tetroxide (Osmic Acid) Fleming's solution Fleming's solution without acetic acid 447. TYPES OF FIXATIVES ACCORDING TO ACTION MICROANATOMIC CYTOLOGICAL CYTOLOGICAL HISTOCHEMICAL (NUCLEAR) (CYTOPLASMIC) 10% formol saline Flemming's Fleming's fuidwithout acetic acid | 10% formol saline 10% neutral buffered formalin Carnoy's, Helly's Abs ethyl alc Heidenhain’s Susa Bouin's Formalin with post chroming Acetone Formal sublimate (formal corsive) Newcomer's Regaud's Newcomer's Zenker’s solution Heidenhain’s Susa Onth's fluid Zenker-Formol (Helly's) Bouin's solution Brasil's solution 448, Factors that affect fixation of tissues: a. RETARDED BY size and thickness of tissue specimens, presence of mucus, presence of fat, presence of blood and cold temperature b. ENHANCED BY size and thickness of tissues and agitation. 449. Air-filled lungs may float on fixative. To avoid this, the organ may be covered with several layers of gauze to maintain it under surface. (Gregorios) 450. ELECTRON MICROSCOPY: The most useful primary fixatives for electron microscopy are osmium tetroxide, glutaraldehyde and paraformaldehyde, with the whole procedure performed at 42C. For routine studies, glutaraldehyde or osmium tetroxide is adequate. For electron histochemistry and electron. immunocytochemistty, Kamovsky's paraformaldehyde-glutaraldehyde is useful. Post-fxation is achieved with osmium tetroxide, 451. Zenker's fluid is made up of mercuric chloride stock solution to which glacial acetic acid has been added just before its use to prevent turbidity ad formation of dark precipitate. It is a good general fixative for adequate preservation of all kinds of tissues, and gives excellent staining results. It is recommended for fixing small pieaces of liver, spleen, connective tissue fibers and nuclei, 452. Zenker-formol (Helly's solution) is an excellent microanatomic fixative for pituitary gland, bone-marrow and blood- containing organs such as spleen and liver. 453. PICRIC ACID FIXATIVES: The yellow stain taken by tissues prevents small fragments from being overlooked. 454. Bouin’s solution is recommended for fixation of embryos and pituitary biopsies.|t is an excellent fixatibe for preserving soft and delicate structures (e.g. endometrial curettings).Page 153 455. Bouin's is NOT suitable for fixing kidney structures, lipid and mucus. It destropys cytoplasmic structures e.g mitochondria 456. Picric acid is highly explosive when dry, and therefore must be kept moist with distilled water or saturated alcohol at 0.5 to 1% concentration during storage. 457. Newcomer's fluid is recommended for fixing mucopolysaccharides and nuclear proteins. It acts both as a nuclear and histochemical fixative. 458. NITRIC ACID is the most common and the fastest decalcifying agent used. DISADVANTAGE: It imparts a yellow color with nitrous acid, thereby impairing the staining reaction of the tissue. This may be prevented by neutralizing the tissue with 5% sodium sulfate and washing in running tap water for at least 12 hours. Addition of 0.19% urea to pure concentrated nitric acid will also make discoloration disappear without considerably affecting the efficiency of the decalcifying solution, 459. Perenyi's fluid consists of nitric acid, chromic acid and ethyl alcohol, it decalcifies and soften tissues at the same time. 460. Von Ebner's fluid consists of HCI, NaCl and water. Itis a moderately rapid decaleifying agent. 461. There are generally four types of tissue impregnation and embedding methods namely: paraffin wax, celloidin (collodion), gelatin and plastic. (Gregorios) 462. MICROTOME ROCKING (CAMBRIDGE) _| For culling serial sections of large block of paraffin emibedded tissues | Paldwell Trefal Simplest among the different types of microtome ROTARY (MINOT) For cuting paraffin embedded sections Minot ‘Most common type used for both routine and research laboratories SLIDING For cutting celloidin embedded sections ‘Adams Most dangerous type of microtome due to movable exposed knife FREEZING For cutting unembedded frozen sections Queckett ULTRATHIN For cuting sections for electron microscopy 463. The cryostat is a refrigerated apparatus used in fresh tissue microtomy, for freezing the tissue into the block holder to the correct degree of hardness to facilitate easier and faster sectioning, It consists of a microtome, usually a rotary microtome, kept inside a cold chamber between -5 to -30°C (average is -20 °C) by an adjustable thermostat, apable of freezing fresh tissues within 2-3 minutes, and cutting sections of 4 u with ease, (Gregoriois GREEN) 464. MICROTOME KNIVES Plane-concave | Less concave sides are recommended for cutting celloidin-embedded tissue blocks on a sliding knife microtome, More concave sides are used to cut paraffin sections on base-sledge, rotary or rocking microtome, Biconcave Recommended for cutting paraffin embedded sections on a rotary microtome. knife Plane-wedge | Recommended for frozen sections or for culling extremely hard and tough specimens embedded in knife paraffin blocks, using a base-sledge type or sliding microtome. 465. APES (3-aminoprpylthriethoxysilane): APES-coated slides are very useful in cytology, particularly for cytospin preparations of proteinaceous or bloody material 466. Infiltration in overheated paraffin (above 60°C) will produce shrinkage and hardening of tissues and destroy lymphoid tissues completely. To avoid this, the paraffin oven must be maintained at a temperature 2 to 5 °C above the melting point of paraffin to be used for impregnation. (Gregorios) 467. Sections are then floated out on a water bath set at 45-50 °C approximately 6-10 °C lower than the melting point of the wax. 468. Peritoneal, pleural and pericardial fluids: jelly-tike clots forming after removal may be prevented by adding 300 units heparin for every 100 ml aspirate. (Gregorios) 469. Gynecologic specimens: Important: sampling of T-zone for detection of dysplasias and carcinomas of the cervix. Transformation zone — junction of endocervial/ ectocervical mucosa. (Gregorios) 470. GYNECOLOGUIC SPECIMENS CYTOLOGIC COLLECTION AND PREPARATION For conventional Pap smear Endocervical brush ‘Samples of endocervical canal ‘Vaginal scrape For patients with hysterectom Lateral vaginal Scrape [[Used for hormonal evaluation Four quadrant vaginal scrape For localization of vaginal adenosis,Page |54 (Wulvar scrape For detection of herpetic lesions or carcinoma 471 472. 473. 474, 475. 476. 477. Equipments’ for vaginal, endocervical and endometrial aspirations (Gregorios RED) a. Glass pipette and rubber bulb for vaginal aspiration b. Ayre’s spatula for swab smear ©. Laryngeal cannula attached to a 10-cc syringe for endocervical or endometrial aspiration To date, Papanicolau (Pap) smear is considered to be the staining method of choice for exfoliative cytology. (Gregorios GREEN). Modified Papanicolau Staining Procedure (Pharr, Wood, Traut). The formula of Pap’s counterstain was changed to produce a more distinct color differentiation between eosinophilic and orangeophilic cytoplasmic stain and omitting Bismarck Brwon from the EA formula. Sharpness of color and brilliant staining reactions are improved due to prepration of counterstain directly from Alcohol, rather than from the aged stock solution of the aqueous stain. (Gregorios) Apoptosis refers to programmed cell death, a normal physiologic process that eliminates unwanted, abnormal or harmful cells. It differs from necrosis, which is the accidental death from trauma, (Rodak) BROKEN SLIDES: Mounting a broken slide on to another xylene-moist slide with a drop of mounting media (Clarite or Permount) may be sufficient for immediate examination while a new section is being cut and stained. If an important slide is broken and replacement is not available, the section (if still intact) may be transferred to another slide. RINGING is the process of sealing the margins of the coverslip to prevent the escape of fluid or semi-fluid mounts and evaporation of mountant to immobilize the coverslip, and to prevent sticking of the slides upon storage. The ringing media used may be Kronig cement made up of two parts paraffin wax mixed with 4-9 parts powdered colophonium resin, heated and filtered. Also available are cellulose adhesives such as Durofix. COUNTERSTAINING Red: Eosin Y, eosin B, phloxine B Yellow: Green: Light green SF. lissamine green icric acid, orange G, rose Bengal Red: Neutral red, safranin O, carmine, hematoxylin Blue: Methylene blue, toluidine blue, celestine blue 478. SPECIAL STAINS CARBOHYDRATES Periodic acid Schiff PAS positive subs - red or magenta red Nuclei — blue Best carmine Glycogen — bright red granules Nuclei ~ blue or grayish blue Mucin, fibrin — weak red Langhan’s iodine method for glycogen (Carleton’s Mod.) Glycogen — mahogany brown Tissue constituents ~ yellow FATS/LIPIDS ‘Sudan IV (Scharlach R) for lipids Lipids (mainly triglycerides = red Nuclei - blue/black Oil red O method in dextrin Fat - biilliant red Nuclei ~ blue ‘Osmic acid stain for fats Nuclei - yellow-orange Fats — black PROTEINS ‘Alkaline fast - green method for basic proteins fones and protamines (found in nuclei) - green Peracetic acid — alcian blue for cystine and cysteine Cystine, cysteine — blue-green ‘Sakaguchi's test for arginine ‘Arginine ~ orange-red NUCLEIC ACID Feuigen technique for nuclear DNA NA — red-purple Cytoplasm = green Methyl green — pyronin method for RNA and DNA DNA (chromatin) — green or blue-green RNA (nucleoli) - rose-red ‘Acridine orange fluorescent staining for RNA and DNA DNA — yellow-green fluorescence RNA — brick to orange redCONNECTIVE TISSUE Page [55 ‘Gomor’s silver impregnation stain for reticulin Reliculin fibers — black ‘Van Gieson’s stain for collagen Collagen — pink or deep red Muscle, cytoplasm , rbe and fibrin ~yellow Nuclei ~ brownish black to black Masson's trichrome stain ‘Weiger'’s elastic tissue stain Verhoef's Muscle, rhc, keratin — red Nuclei — blue-black Collagen, mucus blue Elastic fibers — dark blue or blue-black on clear background Elastic fibers — black Nuclei - gray to black Collagen — red Cytoplasm, muscle ~ yellow ‘Orcein ( Taenzer-Unna-Orcein) Elastic fibers — dark brown Nuclei ~ blue TISSUE PIGMENTS AND DEPOSITS Perl's Prussian blue method for hemosiderin (ferric iron) Hemosiderin and ferric salts - deep blue Tissue, nuclei ~ red (according to counterstain) Masson - Fontana technique of staining melanin and argentaffin cells’ granules Von Kossa’s silver nitrate method for calcium Melanin — black Argentatfin cell granules - black Nuclei ~ red Calcium salts — black Nuclei - red MICROORGANISMS. ‘Wade - Fite for Leprosy bacilli and Nocardia M. leprae, other Mycobact , Nocardia — red Background - blue Nuclei ~ blue-black (if hematoxylin is used) Levaditi's method for spirochetes ‘Spitochetes — black on a yellowish brown background ‘Warthin Starry method for spirochetes Grocott methenamine silver (GMS, mod.) stain for fungi ‘Spirochetes — black Background — golden yellow Fungi — black ‘Mucin, glycogen — gray-black Mycelia, hyphae — old rose rhc — yellow Background ~ pale green 479, The Periodic Acid Schiff (PAS) techniques is the most common method used for the demonstration of basement membrane, particularly the glomerular basement membrane of the kidney due to their CHO content, although electron microscopy seems to be the most informative, (RED Gregorios) 480. AUTOPSY: PRINCIPAL TECHNIQUES a. Technique of R. Virchow ~ Organs are removed one by one. This method has been used most widely, often with ‘some modifications. Originally, the first step was to expose the cranial cavity and from the back, the spinal cord, followed by the thoracic cavity, cervical cavity and abdominal organs b. Technique of C. Rokitansky — This technique is characterized by IN SITU dissection, in part combined with en bloc removal c. Technique of A. Ghon ~ Thoracic and cervical organs, abdominal organs, and the urogenital system are removed ‘as organ blocks. Modification of Ghon’s “EN BLOC” removal technique is now widely used, d. Technique of M. Letulle - Thoracic, cervical, abdominal and pelvic organs are removed EN MASSES and ‘subsequently dissected into organ blocks. 481. For all autopsies, personal protective equipment (PPE) includes scrub suits, gowns, waterproof sleeves, plastic disposable aprons, caps, N95 particulate masks, eye protection (goggles or face shields), shoe covers or footwear restricted to contaminated areas, and double sets of gloves. Cut-resistant and puncture-resistant hand protection (plastic or steel gloves) is also available and certainly recommended for high-risk procedures. (Autopsy Pathology: Manual and Atlas) 482. Primary signs of death: Circulatory failure, respiratory failure and nervous (CNS) failure 483. Secondary signs of death: Algor mortis, Rigor mortis Livor mottis Dessication Putrefaction Postmortem clotting Autolysis. 484, IMMUNOHISTOCHEMICAL TECHNIQUES Used for the IDENTIFICATION OF SPECIFIC OR HIGHLY SELECTIVE CELLULAR EPITOPES OR ANTIGENS in frozen and paraffin-embedded tissuesPage 156 485. Common chromogens for preoxidases are diaminobenzidine (DAB) and aminoethylcarbazole (AEC), both of which should be made fresh immediately before use. (Green Gregorios) 486. EPITHELIAL TISSUE ‘Simple squamous = Forms the serous membranes, or serosae, the slick membranes that ine the ventral cavity epithelium and cover the organs in the cavity ‘Simple cuboidal = Common in glands and their ducts (for example, the Salivary glands and pancreas) epithelium Forms the walls of the kidney tubules and covers the surface of the ovaries ‘Simple columnar = Lines the entire length of the digestive tract from the stomach to the anus epithelium Goblet cells, which produce a lubricating mucus, are often seen in this type of epithelium Pseudostratified = Cells are shorter than others and their nuclei appear at different heights above the basement columnar epithelium ‘membrane + Pseudostratified ciliated columnar epithelium lines most of the respiratory tract; the mucus produced by the goblet cells in this epithelium traps dust and other debris, and the cilia act to propel it upward and away from the lungs Stratified squamous = Found on sites that receive a good deal of abuse or friction, such as the esophagus, the epithelium ‘mouth, and the outer portion of the skin Stratified cuboidal and [+ Both of these epithelia are fairly rare in the body, being found mainly in the ducts of large stratified columnar glands epithe! Transitional epithelium | + Highly modified, stratified squamous pithelium that forms the lining of only a few organs — the urinary bladder. the ureters, and part of the urethra; all of these organs are part of the urinary system and are subject to considerable stratching 487._ GLANDULAR EPITHELIUM Endocrine glands * Lose their connection to the surface (uch), thus they are offen called DUCTLESS GLANDS * Their secretions (all hormones) diffuse directly into the blood vessels that weave through the glands + Examples of endocrine glands include the thyroid, adrenals and pituitary * RETAIN THEIR DUCTS, and their secretions empty through the ducts to the epithelial surface + Include the sweat and oil glands, liver, and pancreas 488. RA 8527: Section 14, Inhibition Against the Practice of Medical Technology.- No person shall practice or offer to practice medical technology as defined in this Act without having previously obtained a valid certificate of registration from the Board provided that registration shall not be required of the following a. Duly registered physicians. b. Medical technologist from other countries called in for consultation or as visiting or exchange professors to college or universities: Provided, they are only practicing the said function. c. Medical technologists in the service of the United States Armed Forces stationed in the Philippines rendering services as such for members of the said forces only. 489. RA 5527: Section 19. Rating in the Examination: In order to pass the examination, a candidate must obtain a general average of at least seventy-five percent in the written test, with no rating below fifty percent in any of the major subjects: Provided, That the candidate has not failed in at least sixty percent of the subjects computed according to their relative weights. 490. RA 8527: Section 23. Refusal to issue Certificate: The Board shall refuse to issue a certificate of registration to any person convicted by the court of competent jurisdiction of any criminal offense involving moral turpitude, or any person guilty of immoral or dishonorable conduct, or of unsound mind, or incurable communicable disease, and in such shall be give to the applicant a written statement setting forth the reason for its action, which statement shall be incorporated in the record of the Board. 491. RA 8527: Section 24. Administrative Investigation - Revocation or Suspension of Certificates: Administrative investigation shall be conducted by at least two members of the Board with one legal officer. No penalty of revocation shall be imposed unless there is a unanimous vote of all the three members of the Board. The Board may, by majority vote, impose the penalty or reprimand or suspension, the latter however not to exceed two years. 492. RA 8527: Section 26. Appeal. — The revocation or suspension of a certificate made by the Board shall be subject to appeal to the Civil Service Commissioner whose decision shall become final thirty days after its promulgation, unless the respondent within the same period has appealed to the office of the President of the Philippines. 493. A.O. No. 2007-0027: Revised Rules and Regulations Governing the Licensure and Regulation of Clinical Laboratories in the Philippines PRIMARY CATEGORY [SECONDARY CATEGORY TERTIARYCATEGORY Routine Hematology ‘Services of primary category laboratory | Services of secondary category Qualitative Platelet Determination Routine Clinical Chemistry laboratory Routine Urinalysis Quantitative Platelet Determination |Special Chemistry Routine Fecalysis ‘Cross-matching — for hospital-based ‘Special Hematology, including Blood Typing — for hospital-based Gram staining - for hospital-based coagulation procedures KOH = for hospital-based Immunology Microbiology — cuiture and sensitivityPage |S7 494. RA 9288: Newborn Screening Act of 2004 Disorder Screened T Effects Screened Effect i Screened and Treated Congenital hypothyroidism ‘Severe mental retardation ‘Normal Congenital adrenal hyperplasia Death Alive and normal Galactosemia Death, cataracis ‘Alive and normal Phenylketonuria ‘Severe mental retardation ‘Normal ‘GEPD deficiency ‘Severe anemia, kericterus ‘Normal 495. DOH Memorandum No, 2012-0164 (May 15, 2012): Inclusion of Maple Syrup Urine Disease (MSUD) in the Newbom Screening Panel of Disorders. MSUD appears to be the most common inborn error of metabolism in the Philippines 496. HIV "Window Period” — refers to the period of time, usually lasting from two weeks to six (6) months during which an infected individual will test “negative” upon HIV testing but can actually transmit the infection. (RA 8504) 497. In 1990, the Philippine Congress enacted the Toxic Substances, Hazardous Wastes and Nuclear Waste Control Act of 1990, commonly known as Republic Act 6969. The Act seeks to protect the public health and environment from unreasonable risks posed by these substances. This legislation is under the jurisdiction of the Department of Environment and Natural Resources (DENR) with the Philippine Nuclear Research Institute (PNRI) as the authority, specific for the control of nuclear/radioactive waste in the Philippines |nttp:/avwaw.fnca.mext.go.jp/english/rwm/news_img/hwm_cr03_07.pdf 498. CODE OF ETHICS As lenter into the practice of Medical Technology, | shall: Accept the responsibilities inherent to being a professional. Uphold the law and shall not participate in illegal work Act in a strict spirit of fairness to all and in a spirit of brotherhood toward other members of the profession. ‘Accept employment from more than one employer only when there in no conflict of interest Perform my task with full confidence, absolute reliability and accuracy. Share my knowledge and expertise with my colleagues, Contribute to the advancement of the professional organization and other allied health organizations. Restrict my praises, criticisms, views and opinions within constructive limits, Treat any information | acquired in the course of my work as strictly confidential Uphold the dignity and respect of my profession and conduct myself @ reputation of reliability, honesty and integrity Be dedicated to the use of clinical laboratory science to promote life and benefit mankind. Report any violations of the above principles of the professional conduct to authorized agency and to the ethics committee of the organization, To these principles, I hereby subscribe and pledge to conduct myself at all times in a manner befitting the dignity of my profession. CODE OF ETHICS As | enter into the practice of Medical Technology, | shall accept the responsibilities inherent to being a professional; | shall uphold the law and shall not engage in illegal work nor cooperate with anyone so engaged; | shall avoid associating or being identified with any enterprise of questionable character; | shall work and act in a strict spirit of faimess to employer, clients, contractors, employees and in a spirit of personal helpfulness and fraternity toward other members of the profession; | shall use only honorable means of competition for professional employment or services and shall refrain form unfairly injuring, directly or indirectly, the professional reputation, projects or business of a fellow medical technologist; | shall ‘accept employment from more than one employer only when there in no conflict of interest; | shall perform professional work in a manner that merits full confidence and trust carried out with absolute reliability, accuracy, faimess and honesty; | shall review the professional work of other medical technologists, when requested, fairly land in confidence whether they are subordinates or employees, authors of proposals for grants or contracts, authors of technical papers or other publications or involved in litigation: | shall advance the profession by exchanging general information and experience with fellow medical technologists and other professionals and by contributing to the work of professional organizations, | shall restrict my praises, criticisms, views and opinions within constructive limits and shall not use the knowledge | know for selfish ends; | shall treat any information | acquired about individuals in the course of my work as strictly confidential, and may be divulged only to authorized persons or entities or with consent of the individual when necessary; | shall report any infractions of these principles of professional conduct to the authorities responsible of enforcement of applicable laws or regulations, or to the Ethics Committee of the Philippine Association of Medical Technologists as may be appropriate. To these principles, | hereby subscribe and pledge to conduct myself at all times in a manner befitting the dignity of my profession,ge [58 499._ PAMET HYMN Beloved PAMET From various lands races and places with grateful hearts we blend our voices This day to our beloved. PAMET from whence unity and love cometh. We join together in brotherhood to live up to thine ideals we should In fields of advancement and learning Thy noble goals maybe our bearing. Loyal and true we'll be to thee Beloved PAMET this we say, for service to God and humanity With joy we sing for thee till eternity. Music: Francis Jerota Pefanco Lyrics: Hector Gentapanan Gayares, Jr. 500._PROFESSIONAL’S OATH |, (NAME) of (RESIDENCE) hereby solemnly swear that | will support and defend the Constitution of the republic of the Philippines; that | will bear true faith an allegiance to the same; that | will obey the laws, legal orders, and decrees promulgated by the duly constituted authorities of the Republic of the Philippines; and that | impose this obligation upon ‘myself voluntarily, without mental reservation or purpose of evasion. | further solemnly swear that at all times and places | will adhere closely to the ethical and professional rules generally accepted by the Medical Technology Professions in the Philippines, and | will well and faithfully discharge to the best of ‘my ability the duties and obligations incumbent upon a legally authorized Medical Technologist. So help me God PANUNUMPA NG PROPESYONAL ‘Ako, si (PANGALAN) ng (TIRAHAN) ay taimtim na nanunumpa na flataguyod ko at ipagtatanggol ang Saligang Batas rg Pilipinas, na akoy tunay na mananalig at tatalima rito, na susundin ang mg utos na legal, at mga atas na ipinahayag ng mga sadyang itinakda ng may kapangyarihan ng Republika ng Pilipinas; at kusa kong babalikatin ang pananagutang ito, na wala ano mang pasubali o hangaring umiwas. Taimtim pa rin akong nanunumpa na sa lahat ng panahon at pook na kinaroroonan, ay mahigpit akong manghahawakan sa mga etikal at tuntuning propesyonal ng mga Medikal Teknolohist sa Pilipinas, at matapat kong gagampanan ng buong husay sa abot ng aking makakaya ang mga tungkulin at pananagutan iniatang sa isang itinakda na Medikal Teknolohist. Kasihan nawa ako ng Diyos DON'T QUIT When care is pressing you down a bit, Rest if you must, but don't you quit.