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Chemical Examination of Urine

Chemical Examination of Urine. Ricki Otten MT(ASCP)SC uotten@unmc.edu. Objectives:. Review the objectives on page 1 and 2 of the lecture handout Objectives marked with ‘*’ will not be tested over during student lab rotation. Historical Perspective: Urinalysis.

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Chemical Examination of Urine

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  1. Chemical Examination of Urine Ricki Otten MT(ASCP)SC uotten@unmc.edu

  2. Objectives: • Review the objectives on page 1 and 2 of the lecture handout • Objectives marked with ‘*’ will not be tested over during student lab rotation

  3. Historical Perspective: Urinalysis • Physical examination of urine • Odor • Taste • Color • Clarity

  4. Historical Perspective • Chemical examination of urine • Limited reactions • Required large volumes of urine • Large volumes of reagent • Performed in test tubes • Time consuming and cumbersome • Clinical usefulness was not realized • Not routinely ordered

  5. Historical Perspective • Microscopic examination of urine • Not until invention of the microscope • Then clinical usefulness realized

  6. Reagent Strip Testing • Technology and necessity • Chemical reactions ‘miniaturized’ • Required less urine • Test results within minutes • Easy to perform • Increased test utilization Brunzel, 2nd Ed, page 124

  7. Reagent Strip Testing • Ideal qualitative screening tool • Sensitive: Low concentration of substances Negative result = normal • Specific: Reacts with only one substance False negative and false positive • Cost effective: Relatively inexpensive tool that provides information about the health status of the patient

  8. Reagent Strip Testing • Chemically impregnated absorbent pads attached to an inert plastic strip • Each pad is a specific chemical reaction that takes place upon contact with urine • Chemical reaction causes the color of the pad to change • Color compared to a color chart for interpretation

  9. Reagent Strip Testing • Qualitative or semi-quantitative results • Concentration units (mg/dl) • Negative, small, moderate large • Negative, 1+, 2+, 3+, 4+ • Timing of chemical reactions is CRITICAL • Shortest time requirement on one end of strip: 30 sec • Longest time requirement on the other: 2 min

  10. Reagent Strip Testing • Principle of chemical reactions • False negative reactions • False positive reactions • Color interferences • Alternative testing: used to confirm results that you may think are invalid due to • Interfering substance • Color interference (called color masking)

  11. Care and Storage (pg 4) Reading assignment: Textbook, chapter 7 Page 124-130 Confirmatory Testing (pg 6)

  12. Confirmatory Testing • Alternative testing establishes the correctness or accuracy of another procedure • Often used when urine is highly pigmented • Bilirubin reagent strip  ictotest

  13. Confirmatory Testing • Characteristics: • Differ in sensitivity • Ictotest vs Bilirubin reagent strip • Differ in specificity • SSA vs Protein reagent strip • Clinitest vs Glucose reagent strip • Differ in methodology/reaction Ideallywant all 3

  14. Differ in Specificity • Clinitest reacts with all reducing substances • Glucose reagent strip reacts with only one reducing substance: glucose

  15. 10 reagent strip tests Specific gravity pH Protein Glucose Ketones Blood Bilirubin Urobilinogen Nitrite Leukocyte Esterase Purpose of the test What is normal What is abnormal Reaction Causes of invalid results

  16. Specific Gravity: Purpose • Evaluates the concentrating and diluting ability of the kidney • Density is related to the amount of substances (solutes) in solution • Increased density ~ increased solute in solution ~ hypertonic urine ~ concentrated urine • Decreased density ~ decreased solute in solution ~ hypotonic urine ~ dilute urine

  17. Specific Gravity: Normal • Normal: 1.002 – 1.035 • Majority of urines: 1.010 – 1.025 • Physiologically impossible: 1.000 >1.040 • Dependent upon hydration status

  18. Specific Gravity: Terms • Isosthenuria • Fixed at 1.010 • Renal tubules lost absorption and secreting capability • Hypersthenuria • Increased specific gravity • Concentrated urine • Hyposthenuria • Decreased specific gravity • Dilute urine Sensitivity issues: Pregnancy testing Urinary tract infection

  19. Specific Gravity: Methods • Methods of measurement • Reagent strip test: indicates ionic solutes • Refractometer: indicates amount of total solutes • Two functions of the kidney • Maintain water balance • Maintain electrolyte homeostasis Performed by renal tubules through concentrating and diluting; reabsorbing and secreting water and electrolytes (ionic)

  20. Specific Gravity: Reaction • Based on a change in the pKa of a polyelectrolyte on the reagent pad • Increased ions in solution causes the polyelectrolyte on the pad to produce free H+ • Free H+ cause a change in pH on the reagent pad • Change in pH: bromthymol blue indicator

  21. Specific Gravity: Reaction

  22. Specific Gravity • Sensitivity: 1.000 • Specificity: detects only ionic substances • Radiographic dye • Mannitol • Glucose Does not interfere

  23. pH: Purpose • Kidneys regulate body’s acid-base balance by selective handling of H+ and HCO3- • Urine pH reflects acid-base status of body • Treatment protocol may require urine pH be maintained at a specific pH (Aids in identification of crystals (microscope))

  24. pH: Normal • Normal: ranges from 4.5 – 8.0 • First morning void: acidic • Physiologically impossible: <4.5 >8.0 • Urine not handled properly • Old urine • Treatment induced

  25. pH: Interpretation • Made in conjunction with • Acid-base status • Renal function • Presence of infection in urinary tract • Diet: high protein, low protein • Medications • Age of urine sample

  26. Acid Respiratory acidosis High protein diet Starvation UTI Alkaline Respiratory alkalosis Vegetarian diet Renal tubular acidosis UTI pH: Abnormal

  27. pH: Reaction • Double indicator system • Methyl red • Bromthymol blue • Amount of free H+ influences acidity of urine and cause pH indicator to change color Needed to measure the wide pH range: acid to alkaline

  28. pH: • Invalid test results due to: • Improper handling of urine sample • Contamination of urine vessel prior to collection • ‘Run-over’ phenomenon

  29. Protein: Purpose • Normal kidneys secrete LITTLE protein <15 mg/dl (or <150 mg/24 hours) • The protein that is found in urine comes from • Bloodstream • Urinary tract • Proteinuria is an indicator of early renal disease • Proteinuria also caused by non-renal disease

  30. Renal Cause of Proteinuria: • Glomerular damage: • Most serious cause of proteinuria • Most common cause of proteinuria • Glomerulonephritis • Nephrotic Syndrome • Tubular dysfunction: • Reabsorption capability decreased • Toxin exposure, inherited disorder • Fancon’s syndrome: heavy metal poisoning

  31. Classification of Proteinuria • Functional • Orthostatic (postural) • Transient • Pathologic • Pre-renal (overflow) • Renal: glomerular • Renal: tubular • Post-renal

  32. Protein: Methods • Reagent strip test • SSA test • Foam test • Micro-albumin test

  33. Protein: Reagent Strip • The reagent pad is held at a constant pH of 3 by a buffer • Proteins (anions) in solution cause an indicator dye to release H+ causing a color change • ‘Protein error of indicators’

  34. Protein: Reagent Strip • Sensitivity: ~ 10-25 mg/dl • Specificity: reacts with albumin • False positive: highly alkaline urine (pH > 8.0) • False negative: Dilute urine Presence of other proteins (Tamm-Horsfall, globulins, myoglobin, free light chains, hemoglobin)

  35. Protein: SSA (Exton’s Test) • Sulfosalicylic Acid (SSA) Precipitation Test • Acid will precipitate proteins out of solution causing the solution to become cloudy • Amount of cloudiness is related to the amount of protein present

  36. Protein: SSA (Exton’s Test) • Amount of cloudiness is evaluated, thus must use centrifuged urine • Sensitivity: 5-10 mg/dl • Specificity: detects all protein

  37. Protein: SSA (Exton’s Test) • False positive results: • Radiographic dyes • Turbid urine • Uncentrifuged urine • False negative results: • Highly alkaline urine • Dilute urine

  38. Protein: Foam test • Shake aliquot of urine and observe color of resulting foam • White foam: protein present

  39. Protein: Micro-albumin test • Measures very low concentration of albumin (better sensitivity than reagent strip test for albumin) • Management of diabetic patient • Methods vary: reagent strip test, immunochemical reaction

  40. Glucose: Purpose • Healthy normal urine does not contain glucose • Normally, glucose is filtered by the glomerulus and is reabsorbed back into the bloodstream through active transport mechanism • Glucose in urine is pathologic

  41. Glucose: Purpose Terms used interchangeably • Glucosuria Glycosuria • Caused by renal and non-renal disease • Pre-renal glycosuria: plasma glucose level exceeds renal threshold (diabetes mellitus) • Renal glycosuria: plasma glucose level below renal threshold, but tubules cannot reabsorb glucose back into bloodstream

  42. Reducing Substances: Purpose • Reducing Substances: • Glucose • Other sugars: galactosemia (inherited metabolic disorder)

  43. Glucose, Reducing Substances • Normal: negative • Abnormal: • Diabetes mellitus: glucose • Impaired renal tubular reabsorption: glucose • Inborn error of metabolism: galactosemia

  44. Methods • Reagent strip: detects only glucose • Copper Reduction: detects reducing substances

  45. Glucose: Reagent Strip • Detects only glucose • Double sequential enzyme reaction

  46. Glucose: Reagent Strip • Sensitivity: ~ 30 mg/dl • Specificity: • Reacts only with glucose • False positive: • Strong oxidizing agents (bleach) • Peroxides • False negative: • Ascorbic acid (reducing agent) • Improperly stored urine: glycolysis

  47. Clinitest Reaction • Copper Reduction Test: • Reducing substances are able to reduce copper sulfate to cuprous oxide • Pass-through phenomenon • All children <2 years: metabolic disorder (galactosemia)

  48. Clinitest Reaction • Sensitivity: ~ 250 mg/dl • Specificity: • Reacts with all reducing substances • Reducing sugars: glucose, galactose, fructose, lactose, maltose (NOT SUCROSE) • False positive: any reducing substance (Ascorbic acid) • False negative: radiographic dye

  49. Ketones are intermediary products of fat metabolism Ketones: Purpose

  50. Ketones • Three ketone bodies • Acetone 2% • Acetoacetic acid 20% • Beta-hydroxybutyric acid 78% • Characteristic ‘fruity breath’ ~ acetone

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