CM PPTs PDF

speed - Tachometer/Strobe light -> RPM P Carry over = Falsely cell count  HAZARDS IN THE LABORATORY “The first step to laboratory safety is to know the hazards insid...

speed - Tachometer/Strobe light -> RPM P Carry over = Falsely cell count  HAZARDS IN THE LABORATORY “The first step to laboratory safety is to know the hazards inside and emanating from the lab.” /Mechanical) Ergonomic Globally Harmonized System (GHS) of Classification and Labeling of Chemicals Includes criteria for the classification of health, physical, and environmental hazards. The standard label elements include: GHS hazard pictograms Signal words GHS hazard statement What to do in case of a chemical spill? Flush the area with large amount of water for 15 minutes, then seek medical attention. Remove contaminated clothing as soon as possible No attempt should be made to neutralize chemicals that come in contact with the skin Proper chemical handling Should not be mixed unless a proper procedure is followed considering the proper order of adding them. Chemical should be used in containers that are easily manageable. NATIONAL FIRE PROTECTION ASSOCIATION HAZARDOUS MATERIAL CLASSIFICATION Classification of Hazards 4: Extreme hazard 3: Serious hazard 2: Moderate hazard 1: Slight hazard 0: No or minimal hazard MATERIAL SAFETY DATA SHEETS 1. Physical and chemical characteristics 2. Fire and explosion potential 3. Reactivity potential 4. Health hazards and emergency first aid procedures 5. Methods for safe handling and disposal CHEMICAL HYGIENE PLAN 1. Appropriate work practices 2. Standard operating procedures 3. PPE 4. Engineering controls (fume hoods, fire safety cabinets) 5. Employee training requirements 6. Medical consultation guidelines 10-20 % O 50-100 mL  Protein-least affected -BaONT URINE PRESERVATIVES § Most routinely used method of preservation § Does not interfere with chemical tests REFRIGERATION § Precipitates amorphous phosphates and urates § Prevents bacterial growth for 24 hours # § Prevents bacterial growth and metabolism BORIC ACID § Interferes with drug and hormone analysis § Keeps pH at about 6.0; Can be used for urine culture transport § Excellent sediment preservative § Acts as a reducing agent interfering with chemical tests for glucose, FORMALIN blood, leukocyte esterase and copper reduction § Rinse specimen container with formalin to preserve cells and casts SODIUM § A good preservative for drug analysis FLUORIDE § Inhibits reagent strip test for glucose, blood and leukocytes URINE PRESERVATIVES § Does not interfere with routine tests; Causes an odor change PHENOL § Use 1 drop per ounce of specimen § Preserves glucose and sediments well THYMOL § Interferes with acid precipitation test for protein § Does not interfere with routine tests TOLUENE § Floats on the surface of the specimen and clings to pipettes and testing materials SACCOMANO § Preserves cellular elements; used for cytological studies FIXATIVE § Composition: 50% ethanol + 2% carbowax (polyethylene glycol) § 10mL of 6N HCl added to 3L to 4L container: for catecholamines, VMA and 5-HIAA collections § Boric acid (1g/dL): preserves urine elements such as estriol and estrogen for up to 7 days § Sodium fluoride: can be added to 24-hour urine for glucose determinations to inhibit bacterial growth and cell glycolysis. TYPES OF URINE SPECIMENS Random Specimen § Most commonly received specimen § Useful for routine screening tests to detect obvious abnormalities First Morning Specimen § Ideal screening specimen; most concentrated § Prevention of false negative pregnancy test and evaluation of orthostatic proteinuria Timed Specimen § Quantitative chemical tests § Measurement of analytes exhibiting diurnal variation § START AND END THE COLLECTION WITH AN EMPTY BLADDER TYPES OF URINE SPECIMENS Catheterized Specimen § Collected under sterile conditions by passing a hollow tube through the urethra into the bladder Midstream Clean Catch Specimen § Provides a safer and less traumatic method of obtaining urine for culture § Alternative to catheterized specimen Suprapubic Aspirate § Collected by external introduction of a needle through the abdomen into the bladder § Preferred sample for bacterial culture and cytological examination TYPES OF URINE SPECIMENS Pediatric Specimen § Done using soft, clear, plastic bags with hypoallergenic skin adhesive to attach to the genital area. § Bag is checked every 15 minutes until the needed amount of sample has been collected.  2 control for bladder or Prostatitis Specimen = Kidney infection C bladder RENAL ANATOMY AND PHYSIOLOGY THE NEPHRON: Functional units of the kidney; 1 to 1.5 million / kidney CORTICAL Make up approximately 85% of nephrons Situated primarily in the cortex Responsible primarily for the removal of waste products and reabsorption of nutrients JUXTAMEDULLARY Have longer loops of Henle that extends deep into the medulla of the kidneys Primarily responsible for urine concentration  PARTS OF THE NEPHRON 1 010. O in  - enclosed by the Bowman's capsule - Renal Compose Glomerulus Site of plasma filtration Proximal Convoluted Major site of tubular reabsorption and tubular secretion Tubule Descending loop of Permeable to water but not to salt; ultrafiltrate Henle increases its concentration Impermeable to water; site of active chloride Ascending loop of Henle reabsorption Site of aldosterone function; retention of sodium in Distal Convoluted Tubule exchange of potassium Collecting Ducts Site of ADH function; retention of water boomL/min  kDA Albumin ↳ intertwining foot processes ↳ -  - - Chorose = 160-180 mylde Ketones = 70 malde Nat = 110-130 minel/ T  Renal Tubular Acidosis O XI I Alkaline = PHCO3 Urine valume  - - Chap vrine volume Cosm -b - = CH2O 2 4 Comox ④ = - ⑰  & O - O O - E D speckled - Providencia Purple = simplest tubular reabsorption test - swollen/Ghost cells crenated  which the of following will contribute the most to specific the gravity using urine strip ? A packed field WBO C. 3+ protein B. many mucus threads D. radiographic contrast media  Total Solids Meter ↓ m 105 - CO -  O - ~ 1-2 drops (9Hs) g a UCOSE -  ⑧ ↓ spar Eromthymal be Pspgr bo ionic strength- Pionic strength ↓ H+ PH + blue yellow Acidic Alkaline  Urine spar determination using a 1 055 refractometer w a reading : - 0 008. 20 of. The specimenstemperature 1027 is C with grams/dl of geocose. - 0 003. 400myld of protein If the 1 : 024 spar of the specimen using - 0 002. the same refractometer is 1035, actual what is the spogr ? ⑱2 - vinem I  CHEMICAL EXAMINATION OF URINE REAGENT STRIPS § Consist of chemical-impregnated absorbent pads attached to a plastic strip § A color reaction takes place when the pad comes in contact with urine § Reactions are interpreted by comparing the color pads with a chart provided by the manufacturer § Results are described as trace, 1+, 2+, 3+, 4+ § 4-parameter: pH, specific gravity, glucose and protein § 10-parameter: pH, sp.gr., glucose, ketones, protein, blood, bilirubin, urobilinogen, nitrite and LE § 11th parameter: Vitamin C (Ascorbic Acid) § 12th / 13th parameter: Creatinine and Microalbumin CARE OF REAGENT STRIPS § Store with desiccant in an opaque, tightly closed container § Store below 30 degrees Celsius, do not freeze § Do not use if chemical pads become discolored § Remove strips from the container just before use QUALITY CONTROL § Test open bottles of reagent strips with known positive and negative controls every 24 hours § Resolve control results that are out of range by further testing § Test reagents used in backup tests with positive and negative controls § Perform positive and negative controls on new reagents and newly opened bottles of reagent strips § Record all control results and reagent lot numbers  § 30 seconds: Bilirubin, Glucose USUAL § 40 seconds: Ketones READING § 45 seconds: Specific Gravity TIME § 60 seconds: pH, Protein, Blood, Urobilinogen, Nitrite § 120 seconds: Leukocyte esterase The amount of time needed for reactions to take place varies between tests and manufacturers, and ranges from an immediate reaction for pH to 120 seconds for leukocytes. For the best semiquantitative results, the manufacturer’s stated time should be followed; however, when precise timing cannot be achieved, manufacturers recommend that reactions be read between 60 and 120 seconds, with the leukocyte reaction read at 120 seconds. I-T Falsely acidic acidic *** To ensure against run-over, blot the edge of the strip with adsorbent paper and holding the strip HORIZONTALLY when comparing it with the color chart. Photometry Rectance -> automated reagent strip reader  fitrable acidity URINE pH ⑨ § Determined based on the concentration of free H+ - § Normal urine pH: 4.5 to 8.0 (random); 5 to 6 (first morning urine) § Most useful parameter for crystal identification § Common reasons for urine pH of greater than 8.0: o Urine specimen that was improperly preserved * o Old urine specimen o Adulterated specimen o Patient was given a highly alkaline substance # PAF MIRL CD55 59 time of day Steep  ACID URINE ALKALINE URINE High protein diet Vegetarian diet Cranberry Citrus fruits Chronic lung disease Renal tubular acidosis D Diabetes mellitus Hyperventilation Dehydration Vomiting Diarrhea Presence of urease-producing bacteria Emphysema - Alkaline tide Escherichia coli Old specimens Ethylene glycol and methanol Starvation UTI Medications URINE PROTEIN § Most indicative of renal disease § Normal urine protein: 2 g/dl (>2000 mg/dL) of sugar is present in urine o Remedy: use two (2) drops of urine instead of five (5) drops ② - ~ - - ~ - O ↳Double sequential STRIP : Ingld reaction enzyme Glucose oxidase CLINITEST = Gugld und ↳ ShS Reducing agent, peroxidase Sol as ↳ - 500 regla URINE KETONES § Results from increased fat metabolism due to abnormal carbohydrate utilization - § Not normally found in urine because metabolized fat is completely broken down into CO2 and H2O § Composition o Β-Hydroxybutyric acid (78%): Cannot be directly measured o Acetoacetic acid (20%): Parent ketone Diacetic acid o Acetone: Measured if glycine is added (2 %) TESTS FOR KETONES 1. Gerhardt’s test § Reaction of ferric chloride with acetoacetic acid forming a port wine or Bordeaux red -- color. § Cannot detect acetone and BHBA 2. Rothera’s test § A nitroprusside ring test § Detect about 1-5 mg/dl of acetoacetic acid and 10-25 mg/dl of acetone. § Cannot detect BHBA - - 3. Hart’s test Indirect method for BHBA detection. § - § Addition of hydrogen peroxide converts BHBA to acetone. § Acetone is then tested by nitroprusside reaction 4. Acetest § Can be used to test urine, serum, plasma and whole blood § 10x more sensitive to acetoacetic acid and acetone, but cannot detect BHBA - § Reagents: o Sodium nitroprusside: main reacting reagent o Glycine: in order for acetone to be measured o Disodium phosphate: buffer o Lactose: for better color differentiation Multistix sodium nitropresside = AAA - chemstrip 1+ glycine = AAA + Actor BLOOD § Urine appearance is dependent on the urine pH, amount and duration of blood in the sample. § Blood should not be detected in the urine under normal conditions § > 5 RBCs / mL: Microscopic hematuria; clinically significant  * Rows stain URINE BILIRUBIN § Yellow pigmented product of hemoglobin degradation § Major clinical significance is for the screening of abnormal hepatobiliary function (Bile duct obstruction) § Bilirubin in the urine is excreted in very small amounts and normally should not be detectable in the urine § Conjugated bilirubin / B2 / Direct bilirubin: bilirubin fraction that can appear in the urine TESTS FOR BILIRUBIN 1. Harrison Spot Test (f) green § Bilirubin is oxidized by ferric chloride in the presence of TCA into biliverdin 2. Ictotest Tablet § More sensitive method (0.05 to 0.10 mg/dL) than the urine strip (0.40 mg/dL) § Less subject to interference § Reagents: o p-nitrobenzene-diazonium-p-toluenesulfonate o Sulfosalicylic acid o Sodium carbonate Afazo reaction o Boric acid § Positive Result: Blue to Purple § If interference in the Ictotest is suspected, it can usually be removed by adding water directly to the mat after the urine has been added. URINE UROBILINOGEN § Colorless product of bilirubin breakdown in the intestines § Appears in the urine as it is filtered by the glomerulus as it passes through the kidneys going to the liver § Excretion peaks at: 2 PM to 4 PM (early afternoon) § Never reported as negative in urinalysis report (< 1 mg/dL or 1 Ehrlich unit) TESTS FOR UROBILINOGEN CHEMSTRIP = Diazo reaction 1.Ehrlich’s reaction > - MASTIX § Uses p-dimethylaminobenzaldehyde and sodium acetate (enhances reaction) § Not specific for urobilinogen (porphobilinogen, indican, sulfonamides, p-aminosalicylic acid, methyldopa, procaine, and chlorpromazine) C) Red 2. Hoesch test Reverse Ehrlich's Test § Uses the Ehrlich reagent dissolved in 6M or 6N HCl (inhibit urobilinogen reaction) § Rapid screening test for urine porphobilinogen 3. Watson Schwartz Differentiation Technique § Classic test for the differentiation of urobilinogen from porphobilinogen § Utilizes organic solvents chloroform and butanol § Interpretation: U B U B U B C U C U C U  PBI O ⑧ cirrhosis, hepatitis , cancer O d never reported as negative URINE NITRITE § Rapid, non-culture method for the detection of UTI § Positive reaction corresponds to: > 100,000 / mL § Most common UTI-causing organisms: Enterobacteriaceae / Coliforms * E. coli, Klebsiella, Proteus, Enterobacter * Nitrosoreductase-positive organisms § Clinical Significance o Cystitis and pyelonephritis o Evaluate antibiotic therapy o Monitor patients at high risk of UTI o Screen urine culture specimens § Factors influencing nitrite result o Organism: nitrate reducing organisms => se Negative o Nitrate: must be present o Duration: 4 hours (1st morning specimen: best) o Antibiotic use: Inhibit bacterial metabolism o Interferences: False-negative 1. Heavy bacteriuria: nitrate à nitrite à nitrogen 2. Vitamin C False-positive 1. Darkly pigmented specimens (bilirubin) LEUKOCYTE ESTERASE Gins/12 seconds § Indicates presence of pyuria and inflammatory process occurring in the kidney or urinary tract § Esterase may be present in: granulocytes, monocytes, macrophages, histiocytes, T. vaginalis § Lymphocytes: WBC that cannot be detected by leukocyte reagent strip § Clinical Significance: tofferedurive/ Highly o Urinary Tract Infection alkaline o Inflammation of the urinary tract Diluted o Screening of urine culture specimens LE + WBCs = none ASCORBIC ACID (11th pad) § A common cause of interference due to its strong reducing property leading to false-negative results for tests utilizing oxidation reaction § In the presence of oxidizers, ascorbic acid is converted to dehydroascorbic acid which is a colorless compound § Compounds such as hydrogen peroxide and diazonium salt are subject to Vitamin C interference § Affects reagents strip reaction for: Blood, Bilirubin, Leukocyte esterase, Nitrite, Glucose False (f BBLNG PRINCIPLES OF REAGENT STRIP TEST FOR ASCORBIC ACID ⑦ Blue 1. C-Stix - § Involves reduction of phosphomolybdates to molybdenum blue by ascorbic acid § Can detect 5 mg/dl of ascorbic acid in the urine after 10 seconds. - 2. Stix and Multistix O § Involves reduction of methylene green by ascorbic acid to its colorless form § Can detect 25 mg/dl of ascorbic acid in 60 seconds. MICROSCOPIC EXAMINATION OF URINE  § The first counting procedure for microscopic sediments of urine § Preservative: Formalin Addis § Normal values count - o Hyaline casts: 0 to 5000 / 12 hours o Red blood cells: 0 to 500,000 / 12 hours o WBCs and epithelial cells: 0 to 1,800,000 / 12 hours PREPARATION AND EXAMINATION OF URINE SEDIMENT § Specimen volume: 10 to 15 mL (12 mL – conical tubes) § Centrifugation: 400 to 500 RCF for 5 minutes § Sediment volume after decantation: 0.5 to 1.0 mL § Sediment volume for examination: 0.02 mL / 20 ul (22 x 22 mm coverslip)  REPORTING Squamous epithelial cells rare, few, moderate, many / LPF Mucus Transitional epithelial cells - Bacteria ↳ Bladder cells Yeast rare, few, moderate, many / HPF Trichomonas - Normal crystals TTBYN Renal Tubular epithelial cells Oval Fat Bodies average number / 10 HPF Red blood cells White blood cells ROWR Abnormal crystals Casts average number / 10 LPF Cast CrAb Spermatozoa present (based on laboratory protocol) PARTS OF A MICROSCOPE adjusted horizontally to adapt to differences in interpupillary distance between Ocular lens operators for optimal viewing conditions * Final / additional / secondary magnification - Adjustment knobs can be rotated to compensate for variations in vision between the operator’s eyes perform the initial magnification Objectives * Initial / Primary magnification - Rheostat regulates light intensity Condenser focuses the light on the specimen Iris Diaphragm controls the amount and the angle of light that will pass through the specimen lens LENS SYSTEM ILLUMINATION SYSTEM § Oculars ↳ § Light source § Objectives Magnification § Condenser system § Adjustment knobs § Iris and aperture diaphragm MICROSCOPIC TECHNIQUES Brightfield § Used for routine urinalysis microscope § Object appears dark against a light background § Aids in Treponema pallidum identification Darkfield § Object appears light against dark background microscope § To adapt from BF: Darkfield condenser § Enhances visualization of elements with low refractive indices. § Works by retardation of light rays diffused by the object in focus, a Phase contrast halo effect is produced around the element, thereby producing better image reinforcement microscope § To adapt from BF: Phase contrast objectives and condenser § DO NOT REQUIRE STAINING MICROSCOPIC TECHNIQUES § Aids in the identification of cholesterol in oval fat bodies, fatty casts and crystals § Used to confirm the identification of fat droplets, oval fat bodies and fatty casts Polarizing § Birefringence: a property indicating that the element can refract light microscope in two dimensions at 90o to each other o Clockwise: positive birefringence o Counterclockwise: negative birefringence § To adapt from BF: Two polarizing filters in crossed configuration § Allows visualization of naturally fluorescent microorganisms or those Fluorescent stained by fluorescent dye microscope § Light source: Halogen quartz lamp MICROSCOPIC TECHNIQUES electron Scanning microscope § Produced a 3D microscopy image § Allows layer by layer imaging of the specimen § Object appears bright against a dark background but without the diffraction halo § Types of interference contrast microscope Interference o Nomarski: differential interference contrast o Hoffman: modulation contrast contrast § To adapt from BF - microscope § Polarizer placed between the light source and the condenser - § Special condenser containing modified Wollaston prisms for each objective - § Wollaston prism placed between the objective and the eyepiece § An analyzer placed behind the Wollaston prism- and before the eyepiece  § Composed of CRYSTAL VIOLET AND SAFRANIN Sternheimer-Malbin § Delineates structure and contrasting colors of the nucleus and (KOVA or Sedi-Stain) cytoplasm § Identifies WBCs, epithelial cells and casts Toluidine Blue § Enhances nuclear details simple stain (Methylene Blue) § Differentiates WBCs and renal tubular epithelial cells § StainsOTAG and neutral fats ORANGE-RED Sudan Dyes § Identifies free fat droplets and lipid containing cells § Differentiates gram (+) and gram (-) bacteria; Identifies Gram stain bacterial casts § Composed of METHYLENE BLUE AND EOSIN Y Hansel Stain § Stains eosinophilic granules; Identifies urinary eosinophils § Stains structures containing iron Prussian Blue Stain § Identifies yellow-brown granules of hemosiderin in cells and (Rous Stain) cast  CELLULAR SEDIMENTS § Squamous Epithelial Cells o Seen in the vaginal lining and lower portions of the male urethra o Largest and most frequently seen and least significant cell in the urine sediment o Central nucleus about the size of an RBC and abundant irregular cytoplasm o CLUE CELLS: Squamous EC infected with G. vaginalis covering most of the cell surface extending beyond the cell’s edges. § Transitional Epithelial Cells (Bladder cells) Urothelial cells o Found lining the renal pelvis, bladder and upper urethra o Spherical, caudate or polyhedral in shape with a centrally located nucleus o Has the ability to reabsorb large quantities of water o No significance in small amounts o Syncytia: increased numbers of transitional cells seen single, in pairs or in clumps; seen after an invasive urologic procedure. § Renal Tubular Epithelial Cells o Lines the renal tubules 10 30 o Round and slightly larger than WBCs with a single, round and eccentrically located nucleus o PCT: Rectangular (Columnar or Convoluted); coarsely granular cytoplasm o DCT: Round or oval and smaller than PCT o Collecting ducts: Cuboidal * o Most significant and indicates tubular injury (>2/HPF), tubular necrosis and renal graft rejection - A neutrophils o Oval Fat Bodies: RTE that contains lipid, found in lipiduria. Exhibit Maltese cross formation. (NEPHROTIC SYNDROME) Smacrophages C transitional EC o Bubble Cells: RTE that contains large nonlipid filled vacuoles (ACUTE TUBULAR NECROSIS) * A squamous EC RED BLOOD CELLS § Swollen: Described as ghost/shadow cells; found in hypotonic urine § Crenated: Shrinking is due to hypertonic urine § Dysmorphic: Seen in glomerular membrane damage; glomerular bleeding § CLINICAL SIGNIFICANCE o Glomerulonephritis o Injury along the genitourinary tract o Renal lithiasis § MOST DIFFICULT TO IDENTIFY! CONFUSED WITH: Yeasts Starch granules Air Bubbles Oil droplets Calcium oxalate crystals Lymphocytes WHITE BLOOD CELLS § Neutrophils purple/violet E+ NBC - None = = o Most frequently seen - o Lyse rapidly in dilute alkaline urine, loss of nuclear detail o GLITTER CELLS: are neutrophils absorbing water in hypotonic urine. Characteristic sparking appearance due to the Brownian movement of their cytoplasmic granules. o LIGHT - BLUE in Sternheimer-Malbin staining (Neutrophils – violet) > § Eosinophils pale de o >1% is considered significant o Highly associated with drug induced interstitial nephritis o May also be increased in UTI and renal transplant § Mononuclear cells o Lymphocytes are the smallest and may resemble RBCs o Monocytes, macrophages and histiocytes are large and may be vacuolated or could contain inclusions CLINICAL SIGNIFICANCE- D o Bacterial conditions (pyelonephritis, cystitis, prostatitis, urethritis) o Nonbacterial conditions (glomerulonephritis, SLE, tumors and interstitial nephritis)  CASTS § Only element found in the urine that are unique to the kidneys § Formed primarily within the lumen of Distal convoluted tubule and Collecting ducts § Formation is favored by urine stasis, acid urine and sodium and calcium presence § Tamm-Horsfall protein or Uromodulin: matrix of cast formation, produced by the RTE § Cylindroids: casts with tapered ends produced at the junction of ALH and DCT § Cylindruria: presence of casts in the urine ORDER OF CAST DEGENERATION Hyaline Casts à Cellular Casts à Granular Casts à Waxy Casts RBC , WBC , RTE ↳ mucus Coarse - Fine Final ↳ uromodelin degeneration products URINE EXAMINATION FOR CAST DETECTION § scanning is performed using the LPO along the edges of the cover slip § observed under subdued light because cast matrix has a low refractive index - § casts are dissolved quickly in dilute alkaline urine § casts must be identified as to composition using the HPO § reported as average # / LPO HYALINE CAST § Only normal and most frequently seen cast § Colorless, homogenous and has same RI with urine; consist almost entirely Tamm Horsfall protein § Nonpathologic: strenuous exercise, dehydration, heat exposure, emotional stress § Pathologic: acute glomerulonephritis, pyelonephritis, chronic renal disease,- congestive heart failure RED BLOOD CELL CAST § Orange-red color, cast matrix containing RBC § Glomerulonephritis, strenuous exercise WHITE BLOOD CELL CAST § Cast matrix containing WBCs § Pyelonephritis, Acute interstitial nephritis (Sterile inflammation) § Presence indicates the need to perform bacterial cultures EPITHELIAL CELL CAST § RTE cells attached to protein matrix § Renal tubular damage &E casts & GRANULAR CAST § Coarse and fine granules in a cast matrix § Glomerulonephritis, Pyelonephritis, stress and exercise Ances = Normal (Hyaline Granular , WAXY CAST § Highly refractile casts with jagged edges and notches § Stasis of urine flow, chronic renal failure FATTY CAST § Fat droplets and oval fat bodies attached to a protein matrix § Nephrotic syndrome, toxic tubular necrosis, diabetes mellitus, crush injuries BROAD CAST § Wider than normal cast matrix; Indicates distention of tubular lumen § Granular and waxy casts are the most common broad casts Renal Failure fasts § Bile-stained broad, waxy casts are seen in tubular necrosis in viral hepatitis § Extreme urine stasis and renal failure  CRYSTALS § Formed from the precipitation of urine solutes (inorganic, organic, medications) § Formation is affected by § pH § Temperature § Solute concentration § Urine pH: most valuable parameter in crystal identification NORMAL CRYSTALS (ACID URINE) urates = yellow-brown calcium = colorless Amorphous urates § Yellow-brown granules (brick dusts) § Pink precipitate on refrigerated specimens (uroerythrin) § Occurs at pH >5.5 § Revert back to uric acid on acidification D Uric acid § Yellow-brown with varying shapes § Confused with cysteine crystals § Highly birefringent on polarized microscopy § Occurs at pH - ↳ haptoglobin is depleted O A. Cystine C. Hematin B. Uric acid D. Radiographic media Pheny Tyrosine n > awine - Failure to observe RBC casts in a urine specimen can be caused by: A. Staining the specimen ~ B. Mixing the sediment after decantation O O C. Centrifuging an unmixed specimen D. Examining the specimen first under low power  Which of the following results are discrepant? A. Small amount of blood but negative protein - - ⑧ O B. Negative blood but 6-10 RBCs/HPF - C. Moderate amount of blood but no RBCs in microscopic exam - D. Negative blood, positive protein - ↳ Intravascular hemolysis  A urine sediment could have which of the following formed elements and still be considered “normal”? O O A. Two or fewer hyaline casts C. A few bacteria B. 5-10 red blood cells D. A few yeast cells Triple phosphate/ammoutnes phosphate  Renal Tubular Epithelial Cells o Lines the renal tubules o Round and slightly larger than WBCs with a single, round and eccentrically located nucleus o PCT: Rectangular (Columnar or Convoluted); coarsely granular cytoplasm o DCT: Round or oval and smaller than PCT o Collecting ducts: Cuboidal o Most significant and indicates tubular injury (>2/HPF), tubular necrosis and renal graft rejection neurochil rophage o Oval Fat Bodies: RTE that contains lipid, found in lipiduria. Exhibit Maltese cross formation. (NEPHROTIC SYNDROME) o Bubble Cells: RTE that contains large nonlipid filled vacuoles SEL (ACUTE TUBULAR NECROSIS) TC RED BLOOD CELLS  Swollen: Described as ghost/shadow cells; found in hypotonic urine  Crenated: Shrinking is due to hypertonic urine  Dysmorphic: Seen in glomerular membrane damage; glomerular bleeding  CLINICAL SIGNIFICANCE Pconc o Glomerulonephritis - o Injury along the genitourinary tract o Renal lithiasis rate  MOST DIFFICULT TO IDENTIFY! CONFUSED WITH: Yeasts Cono Starch granules ↓ Air Bubbles Oil droplets Calcium oxalate crystals ⑧H2O Lymphocytes swell , Ghost cell turomodeli ↳ RBC Coarse > - Fine ↳ WBC muws ↳ pie Urine stasis  Which of the following statements regarding the microscopic examination of urine sediment is false? tumors > AGN, SLE , interstitial nephritis sterile inflammation - O A. If large numbers of leukocytes are present microscopically, then bacteria are present B. If urinary fat is present microscopically, then the chemical test for protein should be positive ↳ Nephrotic syndrome - > massive proteinuria C. If large numbers of casts are present microscopically, then the chemical test for - protein should be positive - D. If large numbers of red blood cells are present microscopically, then the chemical test for blood should be positive - - The following are initial results obtained during a routine urinalysis. Which results should be investigated further? ↓ ↓ A. Negative protein; 0 to 2 hyaline casts - B. Urine pH 6.0; calcium oxalate crystals C. Cloudy yellow urine; specific gravity 1.050 (1 O -. 002 - 1 035) - D. Amber urine with yellow foam; negative bilirubin by reagent - strip; positive Ictotest - -- The test for which of the following results should be repeated? A. Positive blood and protein -phosphate 0- B. pH 5.0, WBCs, and struvite crystals - C. pH 7.0 with ammonium biurate crystals -- D. Positive nitrite and leukocyte esterase The following are initial results obtained during a routine urinalysis. Which results should be investigated further? O A. Negative protein; 2 to 5 waxy casts B. Urine pH 7.5; ammonium biurate crystals C. Cloudy brown urine; 2 to 5 red blood cells D. Clear, colorless urine; specific gravity 1.010 -green is  O Which of the statements regarding examination of unstained sediment is true? O A. Renal cells can be differentiated reliably from WBCs XSyreytig B. Large numbers of transitional cells are often seen after catheterization C. Neoplastic cells from the bladder are not found in urinary sediment X D. RBCs are easily differentiated from nonbudding yeast X Renal Tubular Epithelial Cells o Lines the renal tubules o Round and slightly larger than WBCs with a single, round and eccentrically located nucleus o PCT: Rectangular (Columnar or Convoluted); coarsely granular cytoplasm o DCT: Round or oval and smaller than PCT o Collecting ducts: Cuboidal # o Most significant and indicates tubular injury (>2/HPF), tubular necrosis and renal graft rejection Decoy o Oval Fat Bodies: RTE that contains lipid, found in lipiduria. Is pict Exhibit Maltese cross formation. (NEPHROTIC SYNDROME) # o Bubble Cells: RTE that contains large nonlipid filled vacuoles (ACUTE TUBULAR NECROSIS) Renal tubular epithelial cells are shed in the urine in significant numbers in conditions involving damage to the renal tubules, with malignant renal disease often leading to the highest counts. - -- - - O - X O X C casts  Many stone patients have shown elevation of _____ which is useful as a potential marker for stone disease A. Interleukin 1 B. Complement C3 C. Serum Amyloid A O D. Interleukin 6 - Renal Stone Marker &  S Hunter San Filipo Cetyl trimethyl bromide test y ammonium f white turbidity Acid-albumin test test #) due Metachromatic paper spot =  (Drug-induced) ↳ Hausel stain  Under what urine condition would you observe a swollen RBC? Ghost cell A. Urine pH = 5, and Specific gravity of 1.005 < 1010 O B. Urine pH = 8, and Specific gravity of 1.005 X C. Urine pH = 8, and Specific gravity of 1.025 D. Urine pH = 5, and Specific gravity of 1.025 highly alkaline ileted , Neutrophils o Most frequently seen o Lyse rapidly in dilute alkaline urine, loss of nuclear detail o GLITTER CELLS: are neutrophils absorbing water in hypotonic urine. - Characteristic sparking appearance due to the Brownian movement of their cytoplasmic granules. o LIGHT BLUE in Sternheimer-Malbin staining (Neutrophils – violet) LEUKOCYTE ESTERASE  Indicates presence of pyuria and inflammatory process occurring in the kidney or urinary tract  Esterase may be present in: granulocytes, monocytes, macrophages, histiocytes, T. vaginalis  Lymphocytes: WBC that cannot be detected by leukocyte reagent strip  Clinical Significance: o Urinary Tract Infection o Inflammation of the urinary tract o Screening of urine culture specimens HYALINE CAST  Only normal and most frequently seen cast --  Colorless, homogenous and has same RI with urine; consist almost entirely Tamm Horsfall protein  Nonpathologic: strenuous exercise, dehydration, heat exposure, emotional stress ~Pathologic: acute glomerulonephritis, pyelonephritis, chronic - renal disease, congestive heart failure /hpF  O Impaired ability to reabsorb sodium in PCT: #H O A. Bartter’s syndrome ↓ Nat XLiddle’s syndromePNat C. X B. Gordon’s syndrome P Nat D. Both B and C O   Composed of CRYSTAL VIOLET AND SAFRANIN Sternheimer-Malbin  Delineates structure and contrasting colors of the nucleus (KOVA or Sedi-Stain) and cytoplasm  Identifies WBCs, epithelial cells and casts Toluidine Blue  Enhances nuclear details (Methylene Blue)  Differentiates WBCs and renal tubular epithelial cells  Stains TAG and neutral fats ORANGE-RED Sudan Dyes  Identifies free fat droplets and lipid containing cells  Differentiates gram (+) and gram (-) bacteria; Identifies Gram stain bacterial casts  Composed of METHYLENE BLUE AND EOSIN Y - Hansel Stain  Stains eosinophilic granules; Identifies urinary eosinophils  Stains structures containing iron Prussian Blue Stain  Identifies yellow-brown granules of hemosiderin in cells (Rous Stain) and cast Romanowsky stains - Upon arriving at work, a technologist notices that a urine specimen left beside the sink by a personnel on the night shift has a black color. The specimen has a pH of 6.0 and was sitting - uncapped. What is the most probable cause of the black color? A. Ketones X O - B. Melanin C. Methemoglobin own D. Homogentisic acid X ↳ alkaline  Which of the following sediment constituents does not produce a Maltese cross formation? A. Fatty cast - O B. Bubble cell C. Oval fat body - " D. Starch granule formation Maltese Pseudo cross PARASITES  May be seen in urine sediment due to vaginal or fecal contamination. When noted, repeat examination must be done on a fresh, clean- collected urine specimen.  T. vaginalis: most frequently seen; causes urethral and bladder infection  S. haematobium: excreted together with erythrocytes  E. vermicularis ova : most common contaminant  E. histolytica: reaches the bladder through the lymphatics or fecal contamination  W. bancrofti: accompanied by chyluria (presence of lymph fluid)  PSEUDOCHYLURIA: paraffin-based vaginal creams BROAD CAST  Wider than normal cast matrix; Indicates distention of tubular lumen  Granular and waxy casts are the most common broad casts  Bile-stained broad, waxy casts are seen in tubular necrosis in viral hepatitis  Extreme urine stasis and renal failure -   Composed of CRYSTAL VIOLET AND SAFRANIN Sternheimer-Malbin  Delineates structure and contrasting colors of the nucleus (KOVA or Sedi-Stain) and cytoplasm  Identifies WBCs, epithelial cells and casts Toluidine Blue  Enhances nuclear details (Methylene Blue)  Differentiates WBCs and renal tubular epithelial cells  Stains TAG and neutral fats ORANGE-RED Sudan Dyes  Identifies free fat droplets and lipid containing cells  Differentiates gram (+) and gram (-) bacteria; Identifies Gram stain bacterial casts  Composed of METHYLENE BLUE AND EOSIN Y Hansel Stain  Stains eosinophilic granules; Identifies urinary eosinophils  Stains structures containing iron Prussian Blue Stain  Identifies yellow-brown granules of hemosiderin in cells (Rous Stain) and cast Uric acid crystals - yellow-brown urates es = cna Seen in a variety of shapes, including rhombic, four-sided flat plates (whetstones), wedges, and rosettes. They usually appear yellow-brown but may be colorless and have a six- sided shape, similar to cystine crystals Uric acid crystals are highly birefringent under polarized light, which aids in distinguishing them from cystine crystals Increased amounts of uric acid crystals, particularly in fresh urine, are associated with increased levels of purines and nucleic acids and are seen in patients with leukemia who are receiving chemotherapy, in patients with Lesch-Nyhan syndrome, and sometimes in patients with gout.  REPORTING Squamous epithelial cells Mucus rare, few, moderate, many / LPF I Transitional epithelial cells Bacteria TTBYN Yeast rare, few, moderate, many / HPF Trichomonas Normal crystals Renal Tubular epithelial cells Oval Fat Bodies Red blood cells average number / 10 HPF ROWR White blood cells Abnormal crystals average number / 10 LPF Cast CrAb Casts Spermatozoa present (based on laboratory protocol) CLUE CELLS: Squamous EC infected with G. vaginalis covering most of the cell surface extending beyond the cell’s edges.   Aids in the identification of cholesterol in oval fat bodies, fatty casts and crystals  Used to confirm the identification of fat droplets, oval fat bodies and fatty casts Polarizing  Birefringence: a property indicating that the element can refract microscope light in two dimensions at 90o to each other o Clockwise: positive birefringence o Counterclockwise: negative birefringence  To adapt from BF: Two polarizing filters in crossed configuration  Renal Tubular Epithelial Cells o Lines the renal tubules o Round and slightly larger than WBCs with a single, round and eccentrically located nucleus o PCT: Rectangular (Columnar or Convoluted); coarsely granular cytoplasm o DCT: Round or oval and smaller than PCT o Collecting ducts: Cuboidal o Most significant and indicates tubular injury (>2/HPF), tubular necrosis and renal graft rejection o Oval Fat Bodies: RTE that contains lipid, found in lipiduria. Exhibit Maltese cross formation. (NEPHROTIC SYNDROME) o Bubble Cells: RTE that contains large nonlipid filled vacuoles (ACUTE TUBULAR NECROSIS) Upon examining a urine sample, you notice that the urine has a distinct yellow-brown or greenish color with foam. Which of these crystals would you most likely observe on microscopic examination? Cal OX O Leucine Ammonium birrate  A macroscopic screening of aO red urine sample shows Oturbidity. Which of the following would most likely be found upon microscopic examination? Hematuria A. Yeast and mucus threads B. Bacteria and WBCs ⑧ C. RBCs and RBC casts D. Uric acid crystals and Hyaline casts  A refrigerated urine sample shows amorphous urates after O centrifugation. What should be done to remove the interference before microscopic analysis? A. Add acetic acid to dissolve the crystals B. Perform the analysis without further modification O C. Reheat the sample to 37°C D. Add saline to dilute the specimen acid-o uric acid I rates ° 60 C or below > - dissolved  During a urinalysis, numerous epithelial cells shown in the photo below are observed. What is the most likely cause of their presence? O A. Contamination from poor collection technique B. Recent insertion of catheter into the urinary tract C. Presence of renal tubular necrosis D. Urinary tract infection  A urine sediment analysis shows waxy casts in a patient with renal failure. What is the clinical significance of these findings? A. They indicate good prognosis of the patient’s condition. - O B. They represent end-stage renal disease. C. They are typically found in patients with renal failure. D. They suggest- minimal renal damage. A patient’s urine sample, collected at room temperature, shows the presence of amorphous phosphates. Which urine condition most likely - promotes this crystal formation? X A. Acidic pH and high temperature ④ B. Neutral pH and high specific gravity C. Alkaline pH and low temperature > - white precipitate X D. Acidic pH and low specific gravity M refrigeration   Enhances visualization of elements with low refractive indices.  Works by retardation of light rays diffused by the object in Phase contrast focus, a halo effect is produced around the element, thereby microscope producing better image reinforcement  To adapt from BF: Phase contrast objectives and condenser  DO NOT REQUIRE STAINING oc  When initially focusing on a urine sediment sample using bright-field microscopy, what is a useful strategy to ensure the examination is being performed in the correct plane? A. Focus on any visible crystals O B. Look for an epithelial cell as a reference point X C. Focus on a larger artifact D. Focus on bubbles in the urine  Why is continuous use of the fine adjustment important when examining urine sediment under a microscope? A. It reduces light interference. O B. It helps focus on the correct elements and avoids artifacts. C. It reduces the risk of damaging the slide. D. It automatically identifies sediment constituents. 3 1 2 , , Which of the following statements about oligoclonal bands is false? A. In the CSF, these bands indicate increased intrathecal concentrations of immunoglobulin G - ⑧ B. The bands usually correlate with the stage of disease and can be used to predict disease progression C. The bands are often present in the CSF and serum of individuals with a -- lymphoproliferative disease D. The bands are often present in the CSF but not in the serum of individuals with multiple sclerosis  - Limula horse-shoe crab polyphemis - An unknown fluid can positively identified as being CSF by: A. Determining the lactate concentration B. Determining the albumin concentration C. Determining the presence of oligoclonal banding of electrophoresis O D. Determining the presence of carbohydrate deficient transferrin on electrophoresis Created est  Which of the following CSF tubes is most appropriate for the evaluation of physical characteristics? A. Tube 1 B. Tube 2 O C. Tube 3 D. Tube 2 and 3  200W (e - - - - - - * e G CSF PROTEIN ~ Four CSF tubes were collected, on what tube CSF VDRL is performed? O A. Tube 1 B. Tube 2 and 3 C. Tube 2 O D. Tube 3 and 4 ↳ excess tube -> micro section of priority 0-5(v) -  - Glucose-decreased Glucose-markedly decreased ↓ glose plactate I Val A lactate N  112 of the head 2/3 of the nucleus - 3x5 UM 7 um 45um oor  5447 20 2 0 1 u. 54 , 700 cells) 0) X 1000 sperm concentration =cells/m) 4 , 700 , 000 count 54M X 2m Sperm -  Calculate for the round cell count for a 3 mL semen sample with a sperm concentration of 55 million/mL and 20 spermatids. A. 110 million/mL spermatio sparmland B. 5.5 million/mL O C. 11 million/mL D. 33 million/mL Round cells = Dermatio & NBC Matility = 20UPF Morphology a Locely Seminal vesicle decreased fructose Epididymis decreased neutral α-glucosidase decreased glycerophosphocholine decreased L-carnitine Prostate decreased zinc decreased citric acid decreased glutamyl transpeptidase decreased acid phosphatase Varicocels = most common cause - of male infertility = tapered-head sperm O de O O NSS &