Osmometer 4w6o3m

Laboratory #4

Osmometry

Laboratory #4 Osmolality Skills= 6 Objectives: Upon completion of this exercise, the student will be able to: 1. Define osmol, osmolarity, and osmolality. 2. Know the colligative properties of solutions and what affects them. 3. Explain two (2) methods of determining osmolality of serum or urine. 4. Know the clinical significance of osmolality values. Materials: 1. Advanced Micro-Osmometer- Model 3320 2. 20 µL- Sampler 3. Sampler tips 4. Chamber cleaners 5. Kimwipes 6. 290 mOsm/kg Reference Solution 7. 50 mOsm/kg Calibration Standard 8. 850 mOsm/kg Calibration Standard 9. 2000 mOsm/kg Calibration Standard 10. Controls- two levels Sample requirements: Serum, Plasma, or Urine may be used for testing. Samples should be free of particulate matter. Particulate matter can cause premature crystallization. Centrifuge the sample if turbid. The sample size required for testing is 20 µL. Use the 20 µL pipette to deliver the sample into the sample port. Principle: Osmolality is a measure of the number of dissolved particles of solution. The osmolality is dependent only on the number of particles in solution. The size or charge of the ion or molecule does not affect the measurement. One osmol of a substance is equal to the gram molecular weight divided by the number of particles or ions into which the substance dissociates in solution. Thus, since glucose molecules do not dissociate in aqueous solutions, 1 osmol of glucose = 1 mole = 180 g. For NaCl, which dissociates into 2 ions of aqueous solution, 1 osmol = 58.5 ÷ 2 = 29 grams. A solution containing 1 osmol of solute per kilogram of solvent has a concentration of 1 osmolal. When solutes are dissolved in a solvent, changes occur in the solution’s properties. These changes are referred to as colligative properties, and are dependent on the number of particles in the solution. The colligative properties are: osmotic pressure, 1 MLAB 2401

Osmometry

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vapor pressure, boiling point, and freezing point. The outcome will vary depending on the colligative property affected. 1. 2. 3. 4.

Freezing point is lowered Boiling point is raised Osmotic pressure is increased Vapor pressure is lowered

Because they are in high concentrations in the extracellular fluid and are monoionic, the electrolytes Na+, Cl-, and HCO3-, contribute to over 92% of serum osmolality. Serum proteins, glucose and urea are responsible for the remaining 8%. Many simple formulas have been devised to convert serum solute concentration to osmolality. The formula most easily ed is: Osmolality= 2 (Na) + Glucose + BUN 18 2.8 Although osmolality can be calculated, directly measured values are more significant. Additionally, other analyte values may not be available. Urine osmolality varies depending on the state of hydration. Urine osmolality corresponds well with urinespecific gravity in non-disease states. However, the correlation is poor in renal disease states owing to the greater contribution of high molecular weight substances such as glucose and protein to specific gravity than to osmolality. Osmometry: Osmolality can be estimated by measuring the freezing point depression of a solution. The instrument super-cools (cooled below its freezing point) the solution (either serum, plasma, or urine), in an insulated freezing bath and then mechanical agitation induces crystallization. As crystallization occurs, the heat of fusion increases, causing the sample temperature to rise to a plateau where a liquid/solid equilibrium occurs, which is slightly below the freezing point. The equilibrium or temperature plateau is the freezing point of the specimen. The temperature changes are measured by a thermistor and converted to mosmol/kg H2O. An alternate method for measuring osmolality is the vapor pressure or dew point osmometer. Osmolality measurement in these instruments is indirectly related to a change in vapor pressure, but to the decrease in dew point temperature of the solvent (water) caused by the decrease in vapor pressure of the solvent by the solutes. The sample to be tested is absorbed into a small disk of filter paper which is inserted into a sample holder and then sealed in an enclosed chamber. A temperature sensitive thermocouple is incorporated into the chamber. Initially, the thermocouple is cooled below the dew point. Water in the chamber air condenses and forms a thin film on the surface of the thermocouple. The dew point is the temperature of the atmosphere at which water begins to condense. As water condenses on the junction, heat of 2 MLAB 2401

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condensation is given off. The thermocouple temperature is increased to the dew point temperature, and water ceases to condense. When this temperature is stabilized, it is compared to the initial chamber temperature. This measured temperature change is proportional to the vapor pressure of the evaporating fluid in the filter disk and is calibrated in mosmol/kg H2O by use of known standards. Vapor pressure depression osmometers use smaller volumes of sample than freezingpoint depression osmometers. Another major difference between the two types is that dew point instruments cannot detect the presence of volatiles (alcohols) in solution, whereas freezing point instruments can because volatile solute increases the total vapor pressure of solutions, eliminating the direct solute depression of vapor pressure. Clinical Significance: Osmolality measurements are performed to help evaluate the body’s water balance. Regardless of the particular solute species that is responsible for changes in water balance, an abnormal shift of water between the intravascular and extravascular compartments will result, tending to produce either dehydration or edema. The osmolality will fluctuate as the body responds to the water imbalance.

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Conditions that increase osmolality Serum Urine Dehydration/sepsis/fever/sweating/burns  Dehydration Diabetes mellitus (hyperglycemia)  Syndrome Inappropriate Diabetes insipidus ADH secretion (SIADH)  Adrenal insufficiency Uremia  Glycosuria Hypernatremia  Hypernatremia Ethanol, methanol, or ethylene glycol  High protein diet ingestion Mannitol therapy  Congestive Heart Failure

Conditions that decrease osmolality Serum Urine Excess hydration  Diabetes insipidus Hyponatremia  Excess fluid intake Syndrome Inappropriate ADH  Acute renal insufficiency secretion (SIADH)  Glomerulonephritis

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Laboratory #4

Osmometry

Reporting Results: Reference Values:

Serum/Plasma Urine

275-295 mOsm/kg 50-1200 mOsm/kg

Reportable Range: 0 to 2000 mOsm/kg Linearity:

Linearity is verified every four months using the Five-level linearity set. The linearity set verifies or establishes the reportable range of patient test results. The following conditions should be met: (1) + 3 mOsm/kg between 0-300 mOsm/kg (2)Less than + 1% above 300 mOsm/kg Calibration: Calibration of the Osmometer requires no adjustment by the student. The menu-driven calibration prompts the student to run a series of tests using the calibration standards. When the tests are complete, the Osmometer calculates and stores the calibration coefficients. There are three (3) calibration points: 50, 850, and 2000 Osm/kg. Quality Control: Samples of Clinitrol 290 Reference Solution are to be run daily to check the instrument operation, confirm the calibration, and check repeatability and accuracy of the instrument. The Clinitrol 290 Reference Solution should also be run if erratic results are obtained. Acceptability for the Reference Solution is 290 mOsm/kg + 2. Procedure: 1. The instrument is powered on by pushing the rocker-style power switch on the instrument’s back into the on position. Following the initial display, the student is then prompted to run a two-minute self-diagnostic test. When running the test bar graphs will appear. When the self-diagnostic test is complete, the display will read “Osmometer Ready”. Tests can now be run. 2. Insert a sampler tip into place on the sampler. The sampler tip must be straight and firmly seated. 3. Depress the sampler’s plunger and insert the sampler tip at least 6 mm below the surface of the 290 Reference Standard. Gently release the plunger to load a 20 µL sample. 4. Visually inspect the sample. If there are any large voids or bubbles in the sample, expel the sample and load a bubble-free sample. 5. Wipe the sides of the loaded sampler tip with a Kimwipe to remove any clinging droplets. Then quickly wipe the end of the sampler tip to remove any fluid protruding beyond the tip. Be careful not to “wick” any sample out of the tip. 6. Remove the chamber cleaner from the sample port and discard. Holding the sample by the barrel, insert the tip into the sample port, then rest the sampler in the operating cradle. 4 MLAB 2401

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7. To start the test, push the operating cradle in until it reaches a positive stop. The instrument will run the test for approximately one minute and then display the result. 8. Record the results on the report form. 9. Pull back the operating cradle to a positive stop. 10. Remove the sampler from the operating cradle. 11. Insert a clean dry chamber cleaner into the sample port and rotate it three or four times in both a clockwise and counterclockwise direction. Withdraw the chamber cleaner and insert the opposite end. Rotate the chamber cleaner in the same manner and leave it in the sample port until the next step. 12. Samples will be ran in duplicate. Specimens/Standards, and Controls should repeat + 3 if the value is between 0-400 mOsm/kg; and + 0.75% if the value is 400- 2000 mOsm/kg. The final result will be averaged. 13. Repeat steps 2-7 for the controls and patient sample.

References: Advanced Instruments, Inc., Advanced Micro-Osmometer Operating Manual Model 3320, Advanced Instruments, Inc. 2005

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Laboratory #4

Osmometry

Lab #4: Osmometery Report Form Points = 6 Name:______________ Date: _____________ Instructions: results for 290 Standard, Controls, and Patient. to include units. Each answer, including units, is worth one(1) point.

Standards/Controls/Patient Sample Run 1 290 Standard

Run 2

AVG

C1 C2 Patient name:

Identifier:

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Laboratory #4

Osmometry

Laboratory #4: Osmolality Study Questions Points=14 Name:______________ Date: _____________ Instructions: Each question worth one point unless indicated. 1. Using the course textbook, lab and class notes, define: (1 point each) a. Osmolb. Osmolalityc. Osmolarity-

2. The measurement of osmolality is affected by: a. Size of dissolved particles b. Number of dissolved particles c. Charge of dissolved particles d. Number and charge of dissolved particles 3. Colligative properties of a solution change when the number of solute particles in the solution are changed. Identify the colligative properties and the effect or direction of change when more solute (or particles) are added. (4 points)

4. What is the purpose of the thermistor on the freezing-point osmometer?

5. What are the reference values for serum and urine osmolalities?

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Osmometry

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6. Why are special instruments used to determine osmolality if the value can be calculated from Na, Glucose and BUN values?

7. The type of osmometer currently in use indirectly determines osmolality by measuring which of the colligative properties?

8. What is the clinical significance of osmolality measurement?

9. The calculated osmolality of serum with Na= 139 mmol/L, glucose= 1300 mg/L, and BUN = 140mg/L is:

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