Clinical Microscopy: Urinalysis - PDF Module

Central Philippine University | College of Medical Laboratory Science I. URINALYSIS MEDICAL LABORATORY SCIENCE INTERNSHIP A. Two main types of Urinalysis 1. Dipstick (reagent st...

Central Philippine University | College of Medical Laboratory Science I. URINALYSIS MEDICAL LABORATORY SCIENCE INTERNSHIP A. Two main types of Urinalysis 1. Dipstick (reagent strip urinalysis): commonly MODULE CLINICAL MICROSCOPY performed in screening laboratories and Physician 2 URINALYSIS offices; patient home testing 2. Basic (routine) urinalysis: reagent strip urinalysis + microscopic examination MODULE OUTCOMES B. Two major components of routine urinalysis At the end of this module, the learner should have been able 1. Physicochemical determinations to: a. Appearance 1. discuss comprehensively specimen processing on urine b. Specific gravity with regards to physical, chemical and microscopic c. Reagent strip measurement examination, and other tests. 2. Microscopic examination (Brightfield or Phase 2. correlate correctly physical and chemical urinalysis contrast microscopy) results with microscopic observations and recognize C. Four parts of Routine/Basic Urinalysis discrepancies. 1. Specimen Evaluation 3. know correctly the clinical significance of the results 2. Gross/Physical Examination obtained from the physical, chemical and microscopic 3. Chemical Examination examination of a patient urine sample. 4. Microscopic Examination INTRODUCTION II. SPECIMEN EVALUATION Urinalysis is a set of screening tests used to detect some A. General Information common diseases. It may be used to screen for and/or help 1. Before one proceeds with any examination, the urine diagnose conditions such as urinary tract infections, kidney specimen must be evaluated in terms of its disorders, liver problems, diabetes, or other metabolic acceptability. condition. It involves physical, chemical, and microscopic 2. Each laboratory should have written and enforced examination and also checking the concentration and content guidelines for the acceptance or rejection of of urine. Abnormal urinalysis results may point to a disease specimens. or illness. And it aids in the diagnosis of disease, screening B. Urine Specimen Acceptance asymptomatic populations for undetected disorders, and 1. Properly collected specimen depends on type of monitors the progress of disease and the effectiveness of specimen needed therapy. 2. Properly labelled specimen a. Labels must be attached to the container and not on the lid or on the top of the cover of the container. Clinical Microscopy | MODULE 2 | URINALYSIS 1  b. Labels should not detach to the container even if a. responsible for the pink pigment found in urine its refrigerated due to amorphous deposits especially urates in 3. Quantity is sufficient acidic urine C. Urine Specimen Rejection b. Seen in specimens that have been refrigerated 1. Unlabeled specimen containers 3. Urobilin 2. Specimen does not match the patient’s information a. Imparts an orange-brown color to urine that is 3. Insufficient amount not fresh 4. Improperly transported specimens b. Oxidation product of urobilinogen 5. Contaminated specimen with feces or toilet paper D. Normal Urine Color 6. Containers with contaminated exteriors 1. Pale yellow: dilute random specimen 2. Yellow 3. Dark yellow: concentrated specimens and 1st III. URINE COLOR morning specimen A. General Information 4. Amber 1. Color of urine varies from colorless to black E. Variations of Urine Color 2. Variations may be due to normal metabolic 1. Red urine functions, physical activity, ingested materials or a. Caused by presence of RBC, hemoglobin, and pathologic conditions myoglobin 3. Urine color is mainly determined by its concentration b. Most common cause of abnormal urine color is B. Procedure the presence of blood. 1. Mix urine thoroughly by swirling gently the specimen i. Red is the usual color container. ii. Pink 2. Pour well mixed urine into a test tube and view test iii. Brown from the conversion of hemoglobin to tube against a white background with adequate methemoglobin lighting or c. Nonpathogenic causes of red urine: Examine specimen under a good light source, i. Menstrual contamination in females looking down through the container against a white ii. Ingestion of highly pigmented foods (fresh background beets, blackberries) C. Urine pigments iii. Medications (Rifampin, phenolphthalein, 1. Urochrome phenindione, phenothiazines) a. Responsible for the yellow color of urine d. Urine containing porphyrins may also appear red b. Excretion is generally proportional to the resulting from oxidation of porphobilinogen to metabolic rate porphyrins (port wine color) c. Increased during fever, thyrotoxicosis, starvation e. Differentiation of red urine testing chemically and standing at RT positive for blood 2. Uroerythrin Clinical Microscopy | MODULE 2 | URINALYSIS 2  Urine Color Red Urine i. Green: Ingestion of breath decolorizer (clorets) Urine Transparency Clear Cloudy ii. Blue: Medications (e.g., methocarbamol, methylene blue, amitriptyline) Condition Hemoglobinuria Myoglobinuria Hematuria Appearance of plasma Red Plasma Clear plasma IV. TRANSPARENCY 2. Dark Yellow/Amber/Orange urine A. General Information a. Concentrated urine (Dark yellow) 1. Clarity (Character) is a general term that refers to the b. Dehydration from fever or burns (Amber) transparency/turbidity of a urine specimen c. Bilirubin 2. Clarity provides a key to the microscopic i. Associated with yellow foam when shaken examination results ii. Urine containing bilirubin may contain 3. Amount of turbidity should correspond with the hepatitis virus (Standard Precautions!) amount of material observed under the microscope d. Medications (e.g., Acriflavine, Phenazopyridine, B. Urine Clarity reporting Nitrofurantoin, Phenindione) Term Clarity e. Oxidation of urobilinogen to urobilin Clear No visible particulates seen 3. Brown/Black Few particulates, print easily seen through Hazy a. Additional tests are recommended for urine that urine turns brown/black on standing and have Cloudy Many particulates, print blurred through urine negative test for blood Turbid Print cannot be seen through urine b. Alkaptonuria: urine turns black upon standing Milky May precipitate or be clotted (alkaline urine) because of homogentisic acid C. Nonpathologic turbidity which is a metabolite of phenylalanine 1. Presence of squamous epithelial cells and mucus, c. Melanuria: urine turns black /brown upon particularly in women. standing because melanin an oxidation 2. Bacterial Growth in specimens that are allowed to product of the colorless pigment, melanogen stand. d. Medications (e.g., Levodopa, methyldopa, 3. Precipitation of amorphous phosphates, carbonates, phenol derivatives, argyrols & metronidazole) and urates in refrigerated specimens. 4. Blue/Green urine 4. Semen a. Pathogenic causes 5. Fecal contamination i. Bacterial infections 6. Radiographic contrast media ii. UTI by Pseudomonas species 7. Talcum powder and vaginal creams iii. Intestinal tract infections resulting in D. Pathologic turbidity increased urinary indican 1. Most common causes: RBCs, WBCs, and bacteria b. Non-pathogenic causes 2. Less frequent causes: Nonsquamous epithelal cells, yeast, abnormal crystals, lymph fluid, and lipids Clinical Microscopy | MODULE 2 | URINALYSIS 3  E. Chyluria 2. The specific gravity of a specimen indicates the 1. A rare condition in which the urine contains lymph. relative proportions of dissolved solid components to 2. Associated with obstruction to lymph flow and total volume of the specimen (it reflects the density rupture of lymphatic vessels into the renal pelvis, of the specimen) ureters, bladder, or urethra. 3. Specific gravity is influenced by the number of a. Parasitic infection with Wuchereria bancrofti particles present but also by their size. (filariasis) is the prevailing cause (Even with a. Larger particles, such as proteins and sugars, filariasis, this condition is rare). tend to elevate the specific gravity more than b. Abdominal lymph node enlargement smaller electrolytes. c. Tumors b. Urea contribute more to specific gravity but is 3. The appearance of the urine varies with the amount less value than sodium and chloride, it may be of lymph present (ranges from clear to opalescent or necessary to measure osmolality milky) B. Clinical Correlations F. Lipiduria 1. Sp. gr. of plasma entering the glomerulus is 1.010 1. Presence of fat (fat globules) in the urine a. Hyposthenuric = 1.010 consist of neutral fats (triglycerides) and 2. Normal random specimen = 1.003 -1.035 cholesterol. a. Most random specimens: 1.015-1.025 b. Patients who have sustained skeletal trauma b. 1.035 radiographic contrast media and HMW IV (presumably, the source of lipid is exposed fatty fluid marrow) C. Urinometry c. Exogenous contamination. Oily contaminants 1. Less accurate, not recommended by CLSI such as paraffin may float on the surface of 2. Procedure: urine. a. Urinometer vessel filled ¾ full with urine (10-15 3. Microscopic examination of the urine may be mL). required to classify fatty materials as Oil Red O b. Urinometer inserted with a spinning motion. positive droplets or cholesterol esters with c. Read @ bottom of meniscus. polarization. 3. Correction: a. for every g/dL CHON deduct 0.003 b. for every g/dl glucose deduct 0.004 V. SPECIFIC GRAVITY c. for every 3°C drop in calibration temp. deduct A. General Information 0.001, for every 3°C above the calibration temp. 1. Specific Gravity measures the ability of the kidneys add 0.001. Calibration temp, printed on the to reabsorb essential chemicals and water instrument, usually 20°C Clinical Microscopy | MODULE 2 | URINALYSIS 4  4. Sample problem: A specimen containing 1 g/dL of 2. Testing: protein and 2 g/dL of glucose has a specific gravity a. Urine sample enters a U-shaped glass tube with reading of 1.030. Calculate the correct reading. an electromagnetic coil at one end and a motion 1.030 detector at one end. - 0.003 (protein, 0.003 x 1) b. Electric current applied to the coil causing the - 0.008 (glucose, 0.004 X 2) sound wave to pass through the urine sample. 1.019 c. Frequency is altered by density of the specimen. D. Refractometry d. Microprocessor measures the change in sound 1. Measures specific gravity by measuring refractive wave frequency and coverts the reading to index specific gravity. 2. Refractive index is a comparison of the velocity of G. Falling Drop Method (Dogramaci) light in air with the velocity of light in a solution. 3. More accurate than the refractometer, and is more 3. Procedure: precise than the urinometer. a. Clean surface with a drop of dist. H20 and a 4. Testing: damp cloth, allow to dry. a. This method utilizes a specially designed column b. Put 1-2 drops of sample into the prism. filled with water-immiscible oil. c. Read @ sharp dividing line between light & dark b. A measured drop of urine is introduced into the contrast. column 4. Calibration: c. As this drop falls, it encounters two beams of a. Distilled water: 1.000 light; breaking the first beam starts a timer, and b. 5% NaCl: 1.022 breaking the second turns it off. c. 9% sucrose: 1.034 d. The falling time is measured electronically and is E. Hypersthenuric urine expressed as a specific gravity 1. Specimens with specific gravity readings greater than the refractometer or urinometer scale can be diluted and retested. VI. URINE ODOR 2. Only the decimal portion of the specific gravity is A. General Information multiplied by the dilution factor. 1. Not part of routine urinalysis 3. Example: 2. Seldom of clinical significance but may provide clue Dilution Sp. Gr. reading Actual Sp. Gr. to constituents! 1:2 1.025 1.050 3. Urine normally will have a faint, aromatic odor of 1:2 1.010 1.020 undetermined source. 1:3 1.005 1.015 4. Lack of odor in urine from patients with acute renal F. Harmonic Oscillation Densitometry failure suggests acute tubular necrosis rather than 1. Based on the principle that the frequency of a sound prerenal failure. wave entering a solution changes in proportion to the density of a solution. Clinical Microscopy | MODULE 2 | URINALYSIS 5  B. Common Causes of Urine Odor 6. Reagent strips are also specified by instrumentation Odor Cause manufacturers Aromatic Normal 7. Urine Reagent Strip Parameters Foul, ammonia-like Bacterial decomposition, UTI a. 10 Parameters strip: pH, Protein, Glucose, Fruity, Sweet Ketones Ketones, Blood, Bilirubin, Urobilinogen, Nitrite, Maple Syrup MSUD Leukocytes, and Specific gravity Mousy Phenylketonuria (PKU) b. 4 parameters strip: Specific gravity, pH, Glucose, Rancid Tyrosinemia and Protein Sweaty Feet Isovaleric acidemia c. Other parameters: Ascorbic acid and Creatinine Cabbage Methionine malabsorption 8. Storage of Reagent Strips Bleach Contamination a. Protect from moisture and excessive heat. b. Store in cool, dry area but not in a refrigerator. Rotting Fish Trimethylaminuria c. Check for discoloration with each use; discoloration may indicate loss of reactivity. d. Do not use discolored strips or tablets. VII. CHEMICAL EXAMINATION OF URINE e. Keep container tightly stoppered. A. General Information f. Check manufacturer’s directions with each new 1. Reagent strips are the primary method used for the lot number for changes in procedure. chemical examination of urine. C. Reagent Strip Testing 2. Confirmatory methods are performed when 1. Test urine as soon as possible after receipt. available and necessary. 2. Remove only enough strips for immediate use; 3. Nonreagent strip testing procedures using tablets recap tightly. and liquid chemicals are also available. 3. Test a well-mixed, unspun urine sample. B. Urine Reagent Strips 4. Urine samples must be at room temperature before 1. Reagent strips consists of chemical-impregnated testing. absorbent pads attached to a plastic strip 5. Do not touch the test area with fingers. 2. A color-producing chemical reaction occurs when 6. Do not use reagent strips in the presence of volatile the absorbent pad comes in contact with urine. acids or alkaline fumes. 3. Color intensities are compared on the manufacturers 7. Dip reagent strip into urine briefly—no longer than 1 chart or read by an automated machine. second. iii. Semiquantitative value: trace, 1+, 2+, 3+, 4+ 8. Drain excess urine off—run edge of strip along rim iv. Estimate of the mg/dL concentration of tube, or blot edge on absorbent paper. 4. Two major types of reagent strips are manufactured 9. Do not allow reagents to run together. under the tradenames Multistix (Siemens) and 10. Do not lay reagent strip directly on workbench Chemstrip (Roche) surface. 5. Reagents strips used is a matter of laboratory 11. Follow exact timing recommendations for each preference chemical test. Clinical Microscopy | MODULE 2 | URINALYSIS 6  12. Hold reagent strip close to the color chart, and read 3. The lung excretes carbon dioxide under good lighting. B. Reference Range 13. Know sources of error, sensitivity, and specificity of 1. No normal values assigned to urine pH each test on the reagent strip. 2. In healthy individuals, urine pH may vary from 4.6–8 14. Think! Make correlations between patient history 3. 1st morning: usually acidic, 5-6 and individual test, then follow through. 4. Following meals: alkaline D. Quality Control of Urine Reagent Strips 5. pH of 9.0: improperly preserved specimen 1. Test open bottle of reagent strips with known C. Acid urine positive and negative controls. 1. Diet high in protein and meat 2. When do we run controls? 2. Some fruits, such as cranberries a. Every 24 hours 3. Patient with metabolic or respiratory acidosis should b. When a new bottle of reagent strips is opened produce an acid urine c. When questionable results are obtained 4. Therapeutic acidification d. When there is concern about the integrity of the a. Treat phosphate and calcium carbonate stones strips b. Treat UTI (e.g., methenamine mandelate & 3. Resolve control results that are out of range by fosfomycin tromethamine) further testing. D. Alkaline urine 4. Test reagents used in backup tests with positive and 1. Diet high in fruits and vegetables negative controls. 2. Patient with metabolic or respiratory alkalosis should 5. Record all control results and reagent lot numbers. produce an alkaline urine 6. Several companies manufacture both positive and 3. “alkaline-tide” following a meal negative controls. 4. Induction of alkaline urine (i.e., sodium bicarbonate, 7. Distilled water is not recommended as a negative potassium citrate, and acetazolamide) control a. Treat UA, CaOx or cystine stones 8. All readings of the negative control must be b. used in some UTI’s (the antibiotics neomycin, negative. kanamycin, and streptomycin are more active in 9. Positive control readings must agree with the alkaline urine) published value by ± one color block. c. in sulfonamide therapy d. in the treatment of salicylate poisoning. 5. Clinical Significance VIII. URINE pH a. Respiratory or metabolic acidosis/ketosis A. General Information b. Respiratory or metabolic alkalosis 1. The kidneys and lungs normally work in concert to c. Defects in renal tubular secretion and maintain acid-base equilibrium. reabsorption of acids and bases- renal tubular 2. The kidneys secrete hydrogen in the form of acidosis ammonium ion, hydrogen phosphate and weak d. Renal calculi formation organic acids and reabsorb bicarbonate e. Treatment of UTI Clinical Microscopy | MODULE 2 | URINALYSIS 7  f. Precipitation/identification of crystals e. Usually not detected in a RU g. Determination of unsatisfactory specimens f. Bence Jones Protein E. Summary of pH Reagent Strip i. Found in the urine of persons with multiple Principle Double-indicator system myeloma Reagents Methyl red, bromthymol blue ii. Multiple myeloma is proliferative disorder of Reaction Methyl red + H+ (Red-Orange to immunoglobulin-producing plasma cells. Yellow) → Bromthymol blue – H- iii. Solubility characteristic of BJP: coagulates at (Green to blue) 40°C-60°C and dissolves at 100°C Sensitivity 5 to 9 Remove interference by filtering urine at Sources of error No known interfering substances 100°C Runover from adjacent pads iv. Confirmatory tests for multiple myeloma: Old specimens serum electrophoresis or Correlation with Nitrite immunoelectrophoresis. other tests Leukocytes 2. Renal Proteinuria Microscopic a. Glomerular proteinuria i. Abnormal substances: Amyloid material, toxic substances, and immune complexes IX. PROTEIN ii. Increased pressure from the blood entering A. General Information the glomerulus: Hypertension, strenuous 1. Urinary Protein most indicative of renal disease exercise, and dehydration 2. Proteinuria does not always signify renal disease! iii. Pre-ecclamptic state 3. Normal urine protein: very little b. Tubular proteinuria a. less than 10 mg/dL or 100 mg/24 hours i. Exposure to toxic substances and heavy b. Clinical proteinuria: ≥ 30 mg/dL metals 4. Albumin is the major protein (1/3) in normal urine. ii. Severe viral infections Others are Tamm-Horsfall protein; proteins from iii. Fanconi’s syndrome prostatic, seminal and vaginal secretions iv. Benign proteinuria: Strenuous exercise, high B. Causes of Proteinuria fever, dehydration, and exposure to cold 1. Prerenal proteinuria c. Orthostatic proteinuria a. Aka Overflow proteinuria i. Aka postural proteinuria b. Caused by conditions affecting the plasma prior ii. Persistent benign renal proteinuria frequently reaching the kidney in young adults c. Substances exceed the normal reabsorptive iii. Increased urine protein only when an capacity of the kidneys and appear into the urine individual is in an upright position d. Caused by increased LMW plasma proteins: iv. Proteinuria disappears when a horizontal Hemoglobin, myoglobin, APR’s, and Bence position is assumed Jones protein Clinical Microscopy | MODULE 2 | URINALYSIS 8  v. Caused by increased pressure on the renal Acid buffer vein Reaction Indicator (yellow) + Protein -pH 3.0→ vi. Protein testing: 1st morning urine protein Protein accepts H+ (indicator – H+) negative; urine protein positive after patient Indicator turns blue to green assumes a vertical position for several hours Sensitivity Mulitstix: 15-30 mg/dL albumin d. Microalbuminuria Chemstrip: 6 mg/dL albumin i. Presence of albumin in urine above the Correlation Blood normal level but below the detectable range with other Nitrite of conventional urine dipstick method tests Leukocytes ii. 0-10 mg/dL protein can’t be detected by Microscopic routine reagent strips iii. Monitor patients with DM , hypertension, and D. Protein Strip Reaction Interference peripheral vascular disease 1. False-positive iv. Enables patients to begin treatment before a. Highly buffered alkaline urine (major source of kidney disease occurs error) v. Methods: Reagent strip (Immunochemical b. Pigmented specimens (phenazopyridine) assays) and quantitative detection: c. Quaternary ammonium compounds (detergents) vi. Significant: 30-300 mg albumin/24 hours d. Antiseptics vii. Significant AER: 20-200 ug/min e. Loss of buffer from prolonged exposure of strip 3. Postrenal proteinuria to urine a. Protein is added to the urine as it passes through f. High specific gravity the lower urinary tract (ureters, bladder, urethra, 2. False-negative prostate, and vagina) a. Proteins other than albumin b. Could be caused by b. Microalbuminuria i. Presence of blood (injury or menstrual E. Acid precipitation technique for protein contamination) 1. Precipitates both albumin and globulin ii. Presence of prostatic fluid and large amount 2. Hot precipitation test: used acetic acid or nitric acid of spermatozoa 3. Cold precipitation test: uses ulfosalicylic acid (SSA) iii. Bacterial and fungal infection or Trichloroacetic acid (TCA) C. Summary of Protein Reagent Strip 4. Acetic Acid Test Grading Principle Protein error of indicators Grade Description Reagents Indicator Clear solution; or if turbid before Negative Multistix: tetrabromphenol boiling, no increase in the turbidity. blue Trace Faint turbidity throughout the solution Chemstrip: 3’,3”,5’,5”- Small amount of precipitate filling less 1+ tetrachlorophenol 3,4,5,6 than ¼ of the solution tetrabromosulfopthalein Clinical Microscopy | MODULE 2 | URINALYSIS 9  Moderate amount of precipitate filling 3. Hormonal disorders (e.g., glycogenolysis (glucagon, 2+ from ¼ to ½ of the solution epinephrine, cortisol, thyroxine, and GH) Heavy amount of precipitate filling from 4. Renal glycosuria: glycosuria without hyperglycemia 3+ a. End-stage renal disease ½ to ¾ of the solution 4+ Coagulation of the entire solution b. Cystinosis c. Fanconi’s syndrome F. Albumin: Creatinine Ratio d. Temporary lowering of renal threshold during 5. Provides an estimation of the 24-hr microalbumin pregnancy excretion C. Diabetes Mellitus 6. An albumin: creatinine ratio of 20-30 mg/g is 1. Replace IDDM or juvenile-onset diabetes with "type indicative of albuminuria 1 diabetes" to describe diabetes characterized 7. Albumin reading can be corrected for overhydration primarily by an absolute deficiency of insulin. and dehydration 2. Replace NIDDM or adult-onset diabetes with "type 2 8. Reagent strip reactions: diabetes" to describe diabetes characterized a. Albumin: utilize the dye bis (3’,3”, diodo-4’,4”- primarily by insulin resistance (that is, insulin dihydroxy-5,5”-dinitrophenyl)-3,4,5,6-tetra- ineffective in target tissue) and insulin secretory bromo-sulphonphthalein (DIDNTB) defect. b. Creatinine: principle is based on the 3. "Other specific types" in cases where specific pseudoperoxidase activity of copper-creatinine genetic defects, surgery, drugs, or other things, have complex caused hyperglycemia. 4. Gestational DM (GDM) as a fourth category to describe diabetes that develops during pregnancy. X. URINE GLUCOSE D. Summary of Glucose Reagent Strip A. General Information Double-sequential enzyme reaction 1. Urine glucose is the most frequent chemical analysis Principle (Glucose oxidase) performed on urine Reagents Glucose oxidase 2. Its value is in the detection and monitoring of Peroxidase diabetes mellitus Chromogen: Potassium iodide (green 3. The presence of detectable amounts of glucose in to brown); Tetramethylbenzidine urine is termed glycosuria (yellow to green) 4. Almost all glucose reabsorbed in PCT Reaction Glucose + O2 -Glucose oxidase→ Gluconic 5. Renal threshold is the blood level at which tubular acid +H2O2 reabsorption stops (for glucose, 160-180 mg/dL) H2O +Chromogen -peroxidase→ Oxidized B. Clinical Significance chromogen + H2O 1. Glycosuria often occur in patients with Sensitivity Multistix: 75-125 mg/dL hyperglycemia Chemstrip: 40 mg/dL 2. Diabetes mellitus, gestational diabetes Clinical Microscopy | MODULE 2 | URINALYSIS 10  Correlation Ketones 2+ Larger amount of yellow precipitate; with other Protein yellow green or muddy green mixed tests solution 3+ Larger amount of yellow precipitate; E. Reaction Interference for Glucose Reagent Strip muddy orange mixed solution 1. Glucose oxidase is specific for glucose. 4+ Larger amount of yellow precipitate to 2. False-positive: red precipitate with colorless or light a. Contamination by oxidizing agents (detergents) yellow supernatant; mixed solution may b. Contamination by peroxide be orange to red 3. False-negative: a. High levels of ascorbic acid b. High levels of ketones XI. KETONES c. High specific gravity A. General Information d. Low temperatures 1. The presence of ketones in urine is termed ketonuria e. Improperly preserved specimens (greatest 2. Ketone bodies are the products of incomplete fat source of false-negative results) metabolism. F. Copper Reduction test 3. Increased fat metabolism is caused by: 1. Glucose and other substances reduce cupric ions to a. Inability to metabolize carbohydrates cuprous state in presence of alkali and heat b. Increased loss of carbohydrates CusO4 (Cupric sulfide) + reducing substance -Alkali and c. Inadequate intake of carbohydrates heat → Cu2O (Cuprous oxide) + oxidized substance 4. Ketones testing is most valuable in management (color blue/green to orange/red) and monitoring of patients with type 1 DM 2. Benedict’s solution: copper sulphate, sodium 5. Increased ketones in the blood lead to electrolyte carbonate, sodium citrate buffer imbalance, dehydration, and acidosis and eventual 3. Clinitest tablet: Copper sulphate, sodium carbonate, diabetic coma. sodium citrate, and sodium hydroxide 6. Ketone Bodies 4. Grading of Benedict’s test a. Acetoacetic acid (20%) Grade Description b. Acetone (2%) Negative No change in blue color of reagent or c. Hydroxybutyrate (78%) white green precipitate from phosphate. B. Clinical Significance Also a slight alteration of color without 1. Diabetes acidosis any precipitate. 2. Insulin dosage monitoring Trace Slight amount of yellow precipitate; blue 3. Starvation and green mixed solution 4. Malabsorption/pancreatic disorders 1+ Moderate amount of yellow precipitate; 5. Strenuous exercise apple green mixed solution 6. Vomiting 7. Inborn error of metabolism Clinical Microscopy | MODULE 2 | URINALYSIS 11  C. Summary of Ketone Reagent Strip d. Tumors Sodium nitroprusside e. Trauma Principle (nitroferricyanide) reaction* f. Exposure to toxic chemicals Reagents Sodium nitroprusside g. Anticoagulants Glycine (Chemstrip) h. Strenuous exercise Reaction Acetoacetate (and acetone) + sodium B. Hemoglobinuria alkaline 1. Hemoglobinuria refers to the presence of free nitroprusside + (glycine) - → Purple color hemoglobin in solution in urine Sensitivity Multistix: 5-10 mg/dL acetoacetic acid 2. Occurs when the amount of hemoglobin exceeds Chemstrip: 9 mg/dL acetoacetic acid; haptoglobin 70 mg/dL acetone 3. RBC lysis = clear red urine Interference False positive: phthalein dye, highly a. intravascular hemolysis – no red cells seen in pigmented red urine, Llevodopa, urine medications containing sulfhydryl b. Lysis of red cells in the urine – mixture of groups hemoglobinuria and hematuria False-negative: Improperly preserved 4. Hemosiderin could be found in the RTE and urine specimens sediment. Correlation Glucose a. Hemosiderin are large yellow-brown granules of with other denatured ferritin tests b. Hemosiderin are insoluble form of storage iron; *Does not measure B-hydroxybutyric acid! a product of rbc hemolysis. 5. Clinical Significance of Hemoglobinuria a. Transfusion reactions XII. BLOOD b. Hemolytic anemias A. Hematuria c. Severe burns 1. Hematuria is the presence of an abnormal number d. Infections/malaria of blood cells in urine e. Strenuous exercise/rbc trauma 2. Hematuria is relatively common (hemoglobinuria f. Brown recluse spider bites uncommon, myoglobinuria rare) C. Myoglobinuria 3. Most closely related to disorders of renal or 1. Myoglobinuria is the presence of myoglobin in urine. genitourinary origin. Trauma or damage to the 2. Myoglobin is a heme-containing protein found in organs of these system causes bleeding. muscle tissue. 4. Presence of intact RBC = cloudy red urine 3. Presence of myoglobin is associated with 5. Clinical Significance of Hematuria rhabdomyolysis (muscle destruction) a. Renal calculi 4. Clear red-brown urine b. Glomerulonephritis 5. Heme portion is toxic to the renal tubules = acute c. Pyelonephritis renal failure Clinical Microscopy | MODULE 2 | URINALYSIS 12  6. Clinical Significance of Myoglobinuria F. Reaction Interference for Blood Reagent Strip a. Muscular trauma/crush syndrome 1. False-positive b. Prolonged coma a. Strong oxidizing agents (detergents) c. Convulsions b. Bacterial peroxidases (E. coli) d. Muscle-wasting disease c. Vegetable peroxidases e. Alcoholism/overdose d. Menstrual contamination f. Drug abuse 2. False-negative g. Extensive exertion a. High specific gravity leading to crenated RBCs h. Cholesterol-lowering statin medications (crenated RBCs don’t lyse when they come on D. Hemoglobinuria VS Myoglobinuria contact with reagent pad) Hemoglobinuria Myoglobinuria b. Formalin Color and Clear red- c. Captopril (a hypertension medication) Clear red d. High concentrations of nitrite transparency brown Serum ↑ Bilirubin, ↓ e. Ascorbic acid ↑ CK, LDH f. Unmixed specimen Chemistry haptoglobin Appearance of Red Normal plasma Precipitation XIII. URINE BILIRUBIN Myoglobin not A. Bilirubin with Ammonium Hgb precipitated precipitated 1. A highly pigmented yellow compound sulfate Reagent strip Positive Positive 2. Breakdown product of hgb formed in RES 3. Formation of bilirubin E. Summary of Blood Reagent Strip a. RES: RBC → hgb → heme → protoporphyrin → Pseudoperoxidase activity of biliverdin→B1 Principle hemoglobin b. Circulation: B1 (coupled with albumin) Reagents Peroxide, Tetramethylbenzidine, Buffer transported to liver Reaction H2O2 + chromogen –Hgb Peroxidase→ c. Liver: B1 conjugated with glucoronic acid to form oxidized chromogen (yellow to green to B2. B2 incorporated in bile. green blue color*) + H2O d. Intestines: B2 converted to urobilinogen by Sensitivity Multistix: 5-20 RBCs/mL, 0.015-0.062 intestinal bacteria mg/dL hgb B. Clinical Significance of Bilirubinuria Chemstrip: 5 RBCs/mL, hgb 1. Early indicator of liver disease corresponding to 10 RBCs/mL 2. Conjugated bilirubin appears in the urine when there Correlation o Protein is bile duct obstruction (e.g. gallstones and cancer) with other Microscopic and liver damage (e.g., hepatitis, cirrhosis, and other tests liver disorders) * Intact RBCs produces speckled pattern on pad. 3. Used in determining the cause of jaundice Clinical Microscopy | MODULE 2 | URINALYSIS 13  4. Urine Bilirubin in Jaundice 6. Procedure Condition Urine Bilirubin a. Place 10 drops of specimen on an asbestos- Bile duct obstruction +++ cellulose mat provided with the kit. Liver damage + or - b. Place a reagent tablet on the moistened area of Hemolytic disease - the mat. c. Place one drop of water onto the tablet. Wait 5 C. Summary of Bilirubin Reagent Strip seconds, then place a second drop so that the Principle Diazo reaction water runs off the tablet onto the mat. The tablet Reagents Mulitstix: 2,4-dichloroaniline should be moved to reveal the purple color. A diazonium salt pink or red color is negative. Chemstrip: 2,6-dichlorobenzene- diazonium salt Reaction Bilirubin glucuronide + diazonium salt XIV. URINE UROBILINOGEN acid - → Azodye (tan or pink to violet) A. General Information Sensitivity Multistix: 0.4-0.8 mg/dL bilirubin 1. Bacterial reduction of bilirubin in the intestines: Chemstrip: 0.5 mg/dL bilirubin a. Bilirubin to urobilinogen Interference False positive: highly pigmented i. Some urobilinogen reabsorbed from the urine, Indican, metabolites of Lodine intestines and returned to the liver or filtered False-negative: specimens exposure by the glomerulus. to light*, ascorbic acid, high ii. Urine contains a small amount of concentrations of nitrite urobilinogen (1 mg/dL or Ehrlich unit). Correlation Urobilinogen b. Bilirubin to stercobilinogen with other i. Stercobilinogen not reabsorbed from the tests intestines *Most frequent error is testing of specimens that are not ii. Stercobilinogen oxidized to urobilin (brown fresh. Bilirubin is photo-oxidized to biliverdin! color of feces) D. Ictotest tablet B. Clinical significance 1. Less subject to interference 1. Early detection of liver disease 2. Sensitive than reagent strips 2. Liver disorders (Hepatitis, cirrhosis, carcinoma) 3. Can detect 0.05-0.10 mg/dL bilirubin 3. Hemolytic disorders 4. Requested to detect early stages of liver disease 4. Urine Urobilinogen in Jaundice: 5. Consists of testing mats and tablets Condition Urine Urobilinogen a. p-nitrobenzene-diazonium-p-toluenesulfonate Negative (normal in Bile duct obstruction b. SSA reagent strips) c. Sodium carbonate Liver damage ++ d. Boric acid Hemolytic disease +++ Clinical Microscopy | MODULE 2 | URINALYSIS 14  5. Urine Bilirubin and Urobilinogen in Jaundice False-negative Old specimens, preservation in Urine Urine formalin, high nitrite Condition Bilirubin Urobilinogen Correlation Bilirubin Bile duct Negative (normal with other tests +++ obstruction in reagent strips) *Diazo reaction more specific than Ehrlich reaction! Liver damage + or - ++ D. Ehrlich tube test Hemolytic 1. 1 part Ehrlich reagent + 10 parts urine - +++ disease 2. Postive color: Cherry-red color 3. Subject to interference by porphobilinogen and other C. Urobilinogen Reagent Strip Summary Ehrlich-reactive compounds MULTISTIX E. Watson-Schwartz Differentiation Test Principle Ehrlich reaction 1. Classic test for differentiating urobilinogen and Sensitivity 0.2 mg/dL urobilinogen porphobilinogen Reagents Ehrlich’s reagent 2. Based on solubility differences between (P-dimethylaminobenzaldehyde) urobilinogen and porphobilinogen. Reaction Urobilinogen + Ehrlich’s reagent Chloroform Butanol acid - → red color Urobilinogen soluble soluble False-positive Highly pigmented urine Porphobilinogen insoluble insoluble Ehrlich-reactive compounds (e.g., Other Ehrlich reacting Porphobilinogen, indican, p- cpds (sulfonamides, insoluble soluble aminosalicylic acid, sulfonamides, indican, methyldopa) methyldopa, procaine, chlorpromazine) False-negative Old specimens, preservation in formalin Correlation Bilirubin with other tests CHEMSTRIP Principle Diazo reaction* Sensitivity 0.4 mg/dL urobilinogen F. Hoesch test 1. Rapid screening test for urinary porphobilinogen Reagents Diazonium salt (4- 2. Detects approximately 2 mg/dL porphobilinogen methoxybenzene-diazonium- 3. 2 drops urine + Hoesch reagent.. shake tetrafluoroborate) 4. Hoesch reagent: Ehrlichs rgt + 6M HCl (urobilinogen Reaction Urobilinogen + Diazonium salt - acid is inhibited by the highly acidic pH) → red azodye 5. Positive result: red color False-positive Highly pigmented urine Clinical Microscopy | MODULE 2 | URINALYSIS 15 XV. URINE NITRITE Correlation Protein A. General Information with other tests Leukocytes 1. Rapid screening test for UTI Microscopic 2. Detects bacteriuria E. Reaction Interference for Nitrite Reagent Strip 3. Periodically screen persons at risk for UTI (diabetic 1. False-negative nitrite tests patients, pregnant women) a. Nonreductase containing bacteria 4. Detects initial bladder infection (cystitis) i. Bacteria lacking reductase can’t reduce a. Patients are often asymptomatic nitrate to nitrite. b. Many UTI’s start in the bladder because of ii. Reductase present in gram negative bacteria external contamination that often causes UTI (Enterobacteriaceae) B. Clinical Significance of Urine Nitrite iii. Some gram positive bacteria and yeast does 1. Cystitis not reduce nitrate. 2. Pyelonephritis b. Insufficient contact time 3. Evaluation of antibiotic therapy i. Bacteria must remain in contact with urinary 4. Monitoring of patients at high risk for UTI nitrate long enough to produce nitrite. 5. Screening of culture specimens ii. Specimen for nitrite testing: 1st morning urine C. Reagent Strip Reactions iii. Urine must remain in bladder for 4 hours 1. Results are only reported as positive or negative c. Lack of urinary nitrate 2. Any shade of pink represents a clinically significant d. Green leafy vegetables amount of bacteria e. Large quantities of bacteria converting nitrite to 3. Test does not measure the degree of bacteriuria nitrogen 4. Sensitivity of the test is standardized to correspond f. Antibiotics inhibit bacterial metabolism with a quantitative bacterial culture criterion of g. Ascorbic acid interferes with the Diazo reaction 100,000 organisms per mL. h. Decreased sensitivity in specimens with high D. Nitrite Reagent Strip Summary specific gravity Principle Greiss reaction 2. False-positive nitrite tests Reagents Mulitstix: p-arsanilic acid and a. Improperly preserved specimens Tetrahydrobenzo(h)-quinolin-3-ol i. Multiplication of contaminant bacteria causes Chemstrip: Sulfanilamide and a false (+) result hydroxytetrahydro benzoquinoline acid ii. A true positive nitrite test should be Reaction Aromatic amine + NO2 - → accompanied by a positive leukocyte Diazonium salt + esterase test acid tetrahydrobenzoquinolin - → iii. When fresh urine is used, false-positive Pink azodye results are not obtained, even if a non-sterile Sensitivity Multistix: 0.06-0.1 mg/dL nitrite ion container is used. Chemstrip: 0.05 mg/dL nitrite ion b. Highly pigmented urine Clinical Microscopy | MODULE 2 | URINALYSIS 16  i. Pink discoloration or spotting on the edges of Chemstrip: Indoxylcarbonic acid the reagent pad should not be considered a ester positive reaction! Reaction Acid ester –Leukocyte esterases → indoxyl ii. Automated strip readers report any color + acid indoxyl + diazonium salt change as positive – do visual examination! -acid→purple azodye Sensitivity Multistix: 5-10 WBC/hpf Chemstrip: 10-25 WBC/hpf XVI. LEUKOCYTE ESTERASE Correlation Protein A. General Information with other tests Nitrite 1. LE detects presence of intact and lysed WBCs Microscopic 2. Esterase is present in granulocytic WBCs Longest strip reaction time (2 minutes)! (neutrophils, eosinophils, basophils) and monocytes D. LE Strip Reaction Interference 3. Esterase also present in Trichomonas and 1. False-positive histiocytes a. Strong oxidizing reagents 4. Positive LE test is most frequently accompanied by b. Formalin bacteria! c. Highly pigmented urine (nitrifurantoin) 5. Conditions with leukocyturia without bacteriuria seen 2. False-negative – high concentrations of: in Trichomonas infection, Chlamydia infection, Yeast a. Protein infection, and Interstitial nephritis b. Glucose 6. Chemical tests for leukocytes offers a more c. Oxalic acid standardized detection of leukocytes. Microscopic d. Ascorbic acid examination subject to variation by method used to e. Gentamicin prepare the sediment and technical personnel. f. Cephalosporins 7. Test is not designed to measure WBC g. Tetracyclines concentration. Microscopic examination h. Inaccurate timing recommended. B. Clinical Significance of Urine Leukocytes 1. Bacterial and nonbacterial UTI XVII. SPECIFIC GRAVITY 2. Inflammation of the urinary tract A. General Information 3. Screening of urine culture specimens (LE test more 1. Strip eliminates time consuming step of measuring significant than nitrite test) specific gravity in RU C. LE Reagent Strip Summary 2. Replacing osmometry or refractometry for critical Principle Granulocytic esterase reaction fluid monitoring is not recommended Reagents Diazonium salt B. Clinical Significance Acid ester: 1. Monitoring patient hydration and dehydration Multistix: Deivatized pyrrole 2. Loss of renal tubular concentrating ability amino acid ester 3. Diabetes insipidus Clinical Microscopy | MODULE 2 | URINALYSIS 17  4. Determination of unsatisfactory specimens due to 3. With the usual Western diet, 2–10 mg/dL is excreted low concentration daily, but after ingestion of large amounts of ascorbic C. Urine Specific Gravity Reagent Strip Summary acid, levels in urine may rise to 200 mg/dL. pKa change of polyelectrolytes in an 4. Oxalate and sulfate are the metabolites of ascorbic Principle alkaline medium acid Reagents Bromthymol blue 5. With ingestion of large quantities of ascorbic acid (1 Polyelectrolyte: g or more per day), oxalate stones may form in Multistix: Poly (methyl vinyl susceptible persons. ether/maleic anhydride) B. Significance Chemstrip:Ethyleneglycoldiamino 1. Because of its reducing properties, ascorbic acid ethylethrtetraacetic acid may cause false negative results for: Sensitivity 1.000-1.030 a. Glucose Interference False positive: High concentrations of b. Blood protein c. Bilirubin False-negative: Highly alkaline urine d. Nitrite (>6.5) e. Leukocyte Esterase D. Reagent strip reaction C. Ascorbic Acid Reagent Strip 1. The higher the urine concentration 1. C-Stix (polyelectrolytes), the more H+ are released and a. Testing area is impregnated with absorbed into the pad, lowering the pH on the pad. phosphomolybdates buffered in an acid medium. 2. Bromthymol blue indicator on pad change color from The phosphomolybdates are reduced by blue (alkaline, 1.000) to yellow (acid, 1.030) ascorbic acid to molybdenum blue. 3. Results affected by alkaline pH b. Detects 5 mg/dL of ascorbic acid in urine after 10 4. Add 0.005 to specific gravity readings when the pH seconds is 6.5 or higher. This correction is performed by 2. Stix/Multistix automated strip readers. a. Not as sensitive as C-Stix b. Can detect about 25 mg/dL of ascorbic acid at 60 seconds. XVIII. ASCORBIC ACID c. The reagent is methylene green, which is A. General Information reduced to its colorless form with ascorbic acid. 1. Large quantities of ascorbic acid may occasionally d. Neutral red provides a background color, and the be found in the urine of individuals taking therapeutic overall color changes from blue to purple at doses of vitamin C or other preparations containing levels of 150 mg/dL. abundant ascorbic acid. 2. Urine tests for ascorbic acid have also been used as an indication of adequate ascorbic acid therapy. Clinical Microscopy | MODULE 2 | URINALYSIS 18 XIX. MICROSCOPIC EXAMINATION OF URINE 4. Macroscopic Screening Correlations A. General Information Screening Test Significance 1. Used to detect and identify insoluble materials in Color Blood urine Hematuria VS 2. Must include quantitation of elements present Hemoglobinuria/Myoglobinuria 3. Some elements are considered normal unless Clarity Confirm pathologic or present in increased amounts nonpathologic cause of 4. Least standardized and most consuming type of turbidity routine urinalysis Blood RBCs/RBC casts 5. What the Urine Microscopist must know: Protein Casts/cells a. clinical relevance of urine findings Nitrite Bacteria/WBCs b. chemical abnormalities associated with Leukocyte microscopic interpretations WBCs/WBC casts/bacteria Esterase B. Formed Elements Found in Urine Glucose Yeast 1. Cellular elements a. Desquamated/spontaneously exfoliated D. Factors causing variations in Urine Microscopic epithelial lining cells of the kidney and lower Analysis urinary tract 1. Methods by which the sediment is prepared b. Cells of hematogenous origin 2. Volume of sediment examined 2. Casts 3. Methods and equipment used to obtain visualization 3. Crystals 4. Manner in which results are reported 4. Organisms: bacteria, fungi, viral inclusion cells, and E. Conventional Method Steps parasites 1. Centrifuge urine C. Macroscopic Screening 2. Transfer to glass slide 1. Also known as Chemical sieving 3. Add cover slip 2. Abnormalities in the physical and chemical portions 4. Examine under the microscope of urinalysis play a primary role in the decision to F. Quantitative Counts perform a microscopic analysis. 1. Uses the hematocytometer 3. CLSI recommends that microscopic examination be 2. Uses undiluted well-mixed urine performed when: 3. Normal values: a. Requested by a physician a. Neutrophils = 5–30/μL b. A laboratory-specified population is b. RBCs = 3–20/μL being tested c. Casts = 1–2/μL. c. Any abnormal or physical or chemical 4. Kesson (1978) provided evidence that chamber result is obtained. counts on centrifuged urine sediments are more reliable in predicting renal functional abnormalities than is a conventional method using cells per HPF Clinical Microscopy | MODULE 2 | URINALYSIS 19  5. Addis Count XX. PREPARATION AND EXAMINATION OF THE URINE a. Developed by Addis in 1926 SEDIMENT b. Uses a hematocytometer to count the number of A. Specimen Preparation RBCs, WBCs, casts, and epithelial cells 1. Specimens should be examined while fresh c. Specimen: 12 hour urine 2. Refrigeration may cause precipitation of crystals d. Normal values per 12 hours: 3. Warm specimen to 37ºC to dissolve! i. RBCs = 0-500,000 4. Thoroughly mix the specimen prior to decanting a ii. WBCs & epithelial cells= 0-1,800,000 portion into a centrifuge tube iii. Hyaline cast = 0-5,000 B. Specimen Volume G. Commercial System 1. Standard amount centrifuge usually 10-15 mL; 12 1. KOVA: Hycor Biochemical, Inc. mL frequently used. 2. Urisystem: ThermoFisher Scientific 2. If obtaining a 12 mL specimen is not possible, note 3. Count-10: V-Tech, Inc. specimen volume on report and do correction. 4. Quick-Prep Urinalysis System: Global Scientific Example: if only 6 mL of urine is centrifuged, results 5. Censlide 2000 Urinalysis System: International are multiplied by 2 Remote Imaging Systems C. Centrifugation 6. R/S Workstations 1000, 2000, 2003: DioSys 1. 5 minutes @ 400 RCF H. Sediment Constituents 2. Produces an optimum amount of sediment with least 1. Centrifuged urine sediment should contain all the chance of damage to elements insoluble materials that have accumulated in the 3. RCF = 1.118 X 10-5 X radius in cm X RPM2 urine following glomerular filtration and during 4. Braking mechanism to slow down centrifuge should passage of fluid through the renal tubules and lower not be used urinary tract. 5. All specimens must be centrifuged in capped tubes 2. Formed elements found in urine D. Sediment preparation a. Cellular elements, has two (2) sources: 1. Concentration factor i. Desquamated/spontaneously exfoliated a. Volume of urine centrifuged ÷ sediment volume epithelial lining cells of the kidney and lower b. Used when quantitating the number of elements urinary tract per mL ii. Cells of hematogenous origin 2. Sediment volume frequently used are 0.5 and 1.0 b. Casts mL c. Crystals 3. Resuspend urine sediment in tube prior to d. Organisms: bacteria, fungi, viral inclusion cells, microscopic examination parasites 4. Resuspend by pipette or by tapping the tube with the 3. Sediment constituents may also be classified also finger. Avoid vigorous agitation. into organized sediments (cells, casts, other 5. Urine should be aspirated rather than poured off miscellaneous structures) and unorganized E. Examination of the Sediment sediments (crystals). 1. Volume of sediment examined Clinical Microscopy | MODULE 2 | URINALYSIS 20  a. Glass slide method: 20uL covered by a 22 x 22 4. Lipid stains: Oil red O and Sudan III stains mm cover slip triglycerides and neutral fats orange-red b. Commercial systems: control volume of 5. Gram stain: for differentiation of gram (+) and gram sediment by providing slides with chambers (-) bacteria (always read product literature) 6. Hansel stain: Methylene blue and Eosin Y stains 2. Urine sediment examined in a minimum of 10 LPO eosinophil granules and 10 HPO 7. Prussian Blue stain: stains structures containing iron 3. Slide examined first under LPO, setting changed to (hemosiderin containing cells and casts) HPO for identification H. Cytodiagnostic urine testing 4. LPO scanning of cover-slip perimeter is 1. Not part of Routine urinalysis recommended 2. Slides prepared by cytocentrifugation and stained 5. For detection of casts with Papanicolaou stain 6. Use reduced light when examining unstained 3. Performed by the cytology laboratory sediment by bright-field microscope 4. The urinalysis laboratory should refer specimens F. Reporting the Microscopic Examination with unusual cellular findings to the cytology 1. May vary among laboratories but must be laboratory for further examination. consistent within a particular laboratory system 5. Purpose: 2. Routine Reporting a. For detection of malignancies of the lower Casts per 10 LPFs urinary tract RBC’s and WBC’s per 10 HPFs b. To provide definitive information about renal Epithelial cells, Semiquantitaive terms: tubular changes associated with transplant crystals and other a. 1+, 2+, 3+, 4+ rejection; viral, fungal, and parasitic infections; elements b. Rare, few, moderate, cellular inclusions; pathologic casts; and many inflammatory conditions. 3. Conversion of the number of elements per lpf or hpf to the number per mL provides standardization XXI. MICROSCOPY G. Sediment stains A. Essential Parts of a Microscope 1. Sternheimer-Malbin stain 1. Body a. Most commonly used stain in urinalysis a. Base: supports the microscope and it’s where b. Consists of crystal violet and safranin o: illuminator is located. delineates structure and contrasting colors of the b. Head (Body tube): connects the eyepiece to the nucleus and cytoplasm objective lenses 2. 0.5% toluidine blue: a metachromatic stain; c. Nosepiece (Turret): rotating device that contains enhances nuclear detail the objectives 3. 2% acetic acid: lyses RBCs and enhance nuclei of d. Arm (Stand/Frame): connects the body tube to WBCs the base of the microscope. Clinical Microscopy | MODULE 2 | URINALYSIS 21 2. Illumination system iii. Labeled OIL or HI (homogeneous a. Light source: Lamps/bulbs: Tungsten, Halogen, immersion) or OEL If the objective is Fluorescent, LED; regulated by a Rheostat designed to operate in a drop of oil b. Filters: provide enhanced contrast and color iv. Numerical aperture: designation of the correction (Blue is the most used filter since it amount of light entering the objectives; the absorbs some of the yellow to red light from of greater the numerical aperture, the greater the illumination bulbs) the resolving power c. Condenser: gather the light coming from the light b. Oculars/Eyepiece lens: responsible for further source and to concentrate that light in a resolution (ability to visualize fine details) collection of parallel beams into the specimen; c. Coarse- and fine-adjustment knobs most commonly used (simplest) condenser is the 4. Take note! Abbe condenser a. Iris diaphragm: controls the “amount” of light that d. Iris (Aperture) Diaphragm: contains a number of will pass through the specimen leaves that the operator may open or close to b. Rheostat: controls the “intensity” of light increase the amount of light illuminating the c. Image in the microscope is upside down and object reversed e. Field Diaphragm: may also be present in the light B. Kohler Illumination source; may be opened or closed; used in 1. Koehler illumination is a technique to optimize light focusing the light which passes up through the quality and sharpness by aligning and adjusting condenser each component of the optical system. 3. Lens system 2. Adjustments done to provide optimal viewing of a. Objectives: perform the initial magnification; illuminated objects inscriptions on the objectives are the following: 3. Performed whenever an objective is changed i. Objective magnification: 4×, 10×, 40×, 100× 4. Steps: Objective Ocular Total a. Place a slide on the stage and focus using LPO Objective with the condensers raised Magn. Magn. Mag. Scanning 4× 10× 40× b. Close the field diaphragm LPO 10× 10× 100× c. Lower he condenser until the edges of the field HPO 40× 10× 400× diaphragm are sharply focused d. Center the image of the field diaphragm with the OIO 100× 10× 1,000× condenser centering screws. Total magnification = objective × ocular e. Open the field diaphragm until its image is at the magnification edge of the field ii. Thickness of the cover glass (usually 0.17 f. Remove an eyepiece and look down through the mm) eyepiece tube g. Adjust the aperture diaphragm until approximately 75% of the field is visible Clinical Microscopy | MODULE 2 | URINALYSIS 22  h. Replace the eyepiece ii. 2nd filter (Analyzer): placed between the C. Bright-field Microscopy objectives and the oculars; catches the 1. Most common type of microscopy performed in the refracted light urinalysis laboratory c. Interpretation: 2. Object appear dark against a light background i. Nonbirefringent objects = no refracted light = 3. Sediments must be examined using decreased light no light reaches analyzer = object appears controlled by adjusting the rheostat black 4. Sediment constituents with a low refractive index will ii. Birefringent objects = refracted light = be overlooked when subjected to light with high refracted light reaches analyzer = object intensity appears white/colored against a black D. Phase-Contrast Microscopy background 1. Enhances visualization of elements with low 4. Polarizing microscopy is used in urinalysis to confirm refractive indices, such as hyaline casts, mixed the identification of fat droplets, OFB, and fatty casts cellular casts, mucus threads, and Trichomonas that produces a characteristic maltese cross pattern 2. Eliminates the need to fix or stain living cells F. Interference-Contrast Microscopy 3. Accomplished by adaptation of a bright-field 1. Produces a 3-dimensional microscopy-image and microscope layer-by-layer imaging of a specimen 4. Phase-contrast objective lens contains a phase ring 2. Split the light ray so that the beams pass through that that retards light. As light rays are slowed, there different areas of the specimen. The light is a decrease in the intensity of light producing interference produced by the varied depths of the contrast specimen generates a 3-dimensional image 5. Specific condenser contains also a phase ring with 3. Object appears bright against a dark background a central clear circular area 4. Two types E. Polarizing Microscopy a. Modulation contrast (Hoffman) 1. The use of polarized light aids in identification of b. Differential-interference contrast (Nomarski) birefringent elements: cholesterol on OFB, fatty G. Dark-field microscopy casts, and crystals 1. Used to enhance visualization of specimens not 2. Birefringent: a property indicating that the element viewed with a bright-field microscope can refract light in 2 dimensions at 90 degrees to 2. Aids in identification of Treponema pallidum each other 3. A bright-field microscope is adapted 3. Obtaining a polarized light: a. replacing the condenser with a dark-field a. Brightfield microscopes could be used condenser that contains an opaque disk. b. Two filters installed in a cross formation: b. Disk blocks light from directly entering the i. 1st filter (Polarizing filter): placed in the objective condenser; vibrates light in 1 direction only c. Light rays passes the specimen at oblique reaching the object angles, the light is reflected by the specimen and is captured by the objectives Clinical Microscopy | MODULE 2 | URINALYSIS 23  d. Specimen appears light against a black a. Macroscopic hematuria: advanced glomerular background damage; urinary tract damage (acute infection, H. Fluorescence Microscopy coagulation disorders) 1. Allows visualization of naturally fluorescent b. Microscopic hematuria: early diagnosis of substances or those that have been stained with a glomerular disorders and malignancy of urinary fluorochrome or fluorophore to produce an image tract; renal calculi; nonpathologic (strenuous 2. Fluorescence is the property by which some atoms exercise, menstrual contamination) absorb light at a particular wavelngth and c. renal disease: glomerulonephritis, lupus subsequently emit a light of a longer wavelength nephritis, interstitial nephritis associated with 3. Mechanism: drug reactions, calculus, tumor, acute infection, a. Specimen illuminated with a light of specific tuberculosis, infarction, renal vein thrombosis, wavelength trauma (including renal biopsy), hydronephrosis, b. Excitation filter selects the wavelength of light polycystic kidney, and occasionally acute tubular from the light source (powerful light source necrosis and malignant nephrosclerosis; required: mercury or xenon arc lamps) d. lower urinary tract disease: acute and chronic c. Fluorescent substances absorb the energy and infection, calculus, tumor, stricture, and emit a loner wavelength of light hemorrhagic cystitis following d. Emission filter selects a specific wavelength of cyclophosphamide therapy emitted light from the specimen e. extrarenal disease: acute appendicitis, 4. Fluorescent substances appear as a bright object salpingitis, diverticulitis, acute febrile episodes, against a dark background malaria, subacute bacterial endocarditis, 5. Fluorescent compounds as label polyarteritis nodosa, malignant hypertension, a. Flourescein Isothiocyanate (FITC): green blood dyscrasias, scurvy, and tumors of the emission @ 517nm colon, rectum, and pelvis; b. Tetramethylrhodamine Isothiocyanate f. toxic reactions: drugs, such as sulfonamides, (TRITC): red emission @ 580nm