L3: Basic Techniques

Question 1

Describe why clinical laboratories should monitor water quality.

Answer 1

• directly affects test through constituent reagents, buffers and diluents
• indirectly affects tests through use in washing glassware and autoclaving
• inadequately purified water can cause significant lab error (e.g. calcium from hard water can cause significant error in urine calcium measurements, low grade water could disrupt chromatographic separations due to increase in background noise).

Question 2

Describe parameters to monitor water in clinical laboratories.

Answer 2

There is a need to monitor: 1) ionic, 2) microbial, and 3) organic contamination.

1) Ionic: resistivity is an indicator of ionic contamination (measured in megohm-cm, referenced to 25C) - chlorine should bee removed
2) Microbial: total heterotrophic plate count is an indicator of microbial contamination (CFU/mL). Bacteria may inactivate reagents, contribute to TOC, or alter optical properties of test solution. Alternatively measure epifluorescence microscopy but this is challenging and costly.
3) Organic: total organic carbon (ppb) is an indicator of organic contamination.
4) Particulates and silicates (usually done by the company) can be controlled by including filtration or distillation in the purification system. Silicates or colloidal silica may interfere with some assays.

Question 3

What are the types of water and allowable limits??

Answer 3

Type I (clinical lab reagent water): enzyme/ligand/trace element and heavy metals/quantitative immunofluorescent assays, reagents without preservatives, preparation of standard solutions, electrophoresis, HPLC, tissue/cell culture

Resistivity: 10 megaohm-cm
Microbial content: 10 CFU/mL
Silicate content: 0.05 mg/L
Particulate Matter: >0.22 um

Type II (for preparative techniques): microbiology media preparation, histology stains and dyes, reagents to be sterilized or with preservatives

Resistivity: 1 megaohm-cm
Microbial content: 1000 CFU/mL
Silicate content: 0.1 mg/L
Particulate Matter: N/A

Type II: for glassware washing but not final

Resistivity: 0.1 megaohm-cm
Microbial content: N/A
Silicate content: 1 mg/L
Particulate Matter: N/A

Question 4

Define CLSI water specifications.

Answer 4

1. Clinical laboratory reagent water (CLRW):

resistivity: >100 megaohm-cm
TOC <500 pub
Microbial count <10 CFU/L
Particulate matter using 0.22 um filter

2. Special reagent water: CLRW quality with additional quality parameters and levels defined by the lab to meet the requirements of a specific application
3. Instrument feed water: confirm use of CLRW quality with manufacturer - must meet their specifications.

Question 5

List methods of water purification.

Answer 5

1. Distillation: condensed steam, however it does leave dissolved organic matter
2. Reverse osmosis: semi-permeable membrane - removes particles and dissolved ions
3. Activated charcoal: activated carbon, adsorbs dissolved organic matter, chlorine
4. Deionization: ion exchange resin, binds dissolved ions
5. Ultra-filtration: semi-permeable membrane (>0.22 um) - removes particles and bacteria
6. UV light: oxidation of organic matter (128nm), sterilization (254nm)

1 - > 6 lower capacity, more effective

Question 6

Define normal and reverse osmosis.

Answer 6

Normal osmosis: water and small molecules are able to pass through a semi-permeable membrane but larger molecules are not. If concentration of solutes on each side of the membrane is different, waters flows to the side of higher concentration.

Reverse osmosis: water is forces through a semi-permeable membrane. The process of ion exclusion is a result of the concentration of ions at the membrane surface which forms a barrier that allows other water molecules to pass through while excluding other substances.

Question 7

List the requirements for reverse osmosis.

Answer 7

• carbon pre-filter +/- activated carbon, particularly to remove chlorine which damages the membrane
• sediment pre-filter to remove fine particles which will clog the membrane
• periodic back flushing of the system is necessary to prevent the formation of scale on the membrane
• harness reduction in areas with hard water (Ca precipitates out)

Question 8

Describe Quality Assurance requirements for water.

Answer 8

• Clinical laboratories should have a QA program to monitor water quality
• Must be measured frequently enough to detect potential changes in the system
• Look for trends to anticipate maintenance before water quality degrades
• Components fail: 1) UV lamps deteriorate with use, 2) ion-exchange or sorption beds become exhausted with contaminates, 3) filters can become blocked, perforated, or contaminated

Question 9

Describe Quality Assurance requirements for water.

Answer 9

• Clinical laboratories should have a QA program to monitor water quality
• Must be measured frequently enough to detect potential changes in the system
• Look for trends to anticipate maintenance before water quality degrades
• Components fail: 1) UV lamps deteriorate with use, 2) ion-exchange or sorption beds become exhausted with contaminates, 3) filters can become blocked, perforated, or contaminated

Question 10

List clinical utilities of centrifugation.

Answer 10

1. Specimen processing: serum and plasma separation from clots/cells, concentration of cellular/particulates for microscopy (small bench top)
2. Analytical: liquid-to-liquid extraction (partitioning), washing suspension (solid phase particles), ultra-filtration (free drug/hormone analysis), gradient density centrifugation (lipids) - larger capacity
3. Trouble shooting: removing interferences and precipitates (ultra-centrifuge)

Question 11

Define relative centrifugal force (RCF).

Answer 11

force required to separate two phases.

Expressed as number of times greater than gravity (xg).
RCF= 1.118x10^-5 x r x rpm^2
r= radium (cm) to the sample
rpm: revolutions per minute

When given rpm and not RCF, radial dimensions or rotor type need to be given.

Question 12

What are swinging bucket rotors?

Answer 12

• buckets start vertical, swing into position
• as buckets swing outward, particles travel in a constant manner along the tube at right angles to the shaft
• creates a well-packed pellet
• maximum force <6500 xg

Question 13

What are fixed angle rotors?

Answer 13

• tubes stay at constant angles with respect to the axis of rotation
• generate less heat, can go faster than swing bucket style
• particles collide with the walls of the tube and the settle at the bottom
• max speed <60,00 xg - used for collecting microorganisms, cellular debris, large organelles, precipitated organelles,
• ultracentrifuges are often fixed angle with maximal speeds of 100,000-600,000 xg (used for lipid fractionation, sedimentation analysis etc

Question 14

Describe QA processes for centrifuges.

Answer 14

Daily: preventative maintenance (cleaning, visual checks)
Monthly: functional verification (timer, speed, lubricant, tachometer)
Annually: service maintenance by Hospital Biomedical Engineering or an external service provider.

Accreditation requires centrifuges are cleaned and maintained, there is record of maintenance, speed should bee checked at least annually.

Question 15

Define osmometry, osmolarity, and osmolality.

Answer 15

Osmometry: measure of osmotic pressure

Osmolarity: measure of osmoles of solute per litre of solution (mol/L). Since the volume of solution changes with the amount of solute added as well as changes in temperature and pressure, osmolarity is difficult to determine.

Osmolality: measure of osmoles of solute per kg of solvent (mol/kg). Since the amount of solvent will be constant, osmolality is easier to evaluate. Commercially available osmometers report results using osmolality units mOsm/kg

Question 16

Define colligative properties.

Answer 16

When solutes are dissolved in a solute, the colligative properties of the solution change in a linear relationship as the number of particles in solution increases.

Increased osmolality affects solvents by:

• increasing boiling point and osmotic pressure
• decreasing freezing point and vapour pressure

We can determine the amount of solute in a sample by measuring any one of these properties.

Question 17

Define osmometry and list main types of osmometers.

Answer 17

osmometry: measure of the osmotic strength of a solution, colloid, or compound
1. freezing point osmometers: determine the osmotic strength of solution by utilizing freezing point depression
2. vapour pressure osmometers: determine the concentration of osmotically active particles that reduce vapour pressure of the solution.
3. membrane osmometers: measure of the osmotic pressure of a solution separated by a semi-permeable membrane (not very popular due to variability in membrane types and clogging issues)

Question 18

Describe freezing point osmometry (FPO).

Answer 18

• A solution is supercooled several degrees below its freezing point and then mechanically agitated to partially crystallize
• The heat of fusion is suddenly liberated and the sample temperature rises to a plateau. Here, a temperature equilibrium between the solid and liquid phases occurs and represents the freezing point of the solution
• The addition of 1 most of solute into 1 kg of water will cause a decrease of 0.000185C in freezing point
• thermistor probe connected to a circuit can very precisely measure the heat of fusion to determine the concentration
• typical lab measures 0-2000 most/kg

Question 19

List advantages and disadvantages of freezing point osmometry.

Answer 19

Advantages:

• performs rapid and inexpensive measurements
• simple and reliable performance
• industry preferred method
• small sample size
• ideal for dilute biological and aqueous solutions

Disadvantages:

• samples must be of low viscosity
• not ideally suited for high molality or colloidal solutions

98% of labs use this!

Question 20

Describe vapour pressure osmometry.

Answer 20

Determine the concentration of osmotically active particles that reduce vapour pressure of the solution. Similar to FPO, you place a sample and you look at VP and then you compare it to reference material. The decrease in VP is proportional to the increase in osmolality.

Question 21

List advantages and disadvantages of vapour pressure osmometry.

Answer 21

Advantages:

• performs rapid and inexpensive measurements
• small sample size
• ideal for dilute and aqueous solutions

Disadvantages:

• less precise than FPO
• dependent on ambient temperature
• cannot be used for volatile solutes like alcohols (escape and therefore raise rather than lower vapour pressure)
• not ideal for high molality or colloidal solutions

Question 22

Define plasma osmolality and the osmolal gap. List clinical examples.

Answer 22

Plasma osmolality can be estimated by calculating 2[Na]+[glucose]+[urea] in mol/L

Calculated and measured osmolality differ due to other solutes, this difference is called the osmolal gap

Normally, measured plasma osmolality is 280-3000 mOsm/kg. If the gap exceeds 10, a significant amount of other solutes are “hidden” in the plasma

The osmolal gap is widely used to screen for alcohol poisoning since ethanol will increase the osmolality of plasma.

Question 23

Define urine osmolality and osmolar gap and provide clinical examples.

Answer 23

Assessed the urine concentrating ability of the kidney since normal is 50-1200 mOsmkg

Urine osmolality can be estimated by calculating: 2([Na]+[k])+ urea

Gap sometimes used to estimate ammonia, but this is not common. Another example is urine dipstick testing.

Question 24

Describe urine dipstick testing and specific gravity.

Answer 24

Specific gravity is an index of urine solute concentration but is affected by # and weight of solutes; neutral and high MW solutes (glucose and proteins) are not detected by dipstick screening

Dipstick is sensitive to ions, Na, K displaces H and thus changes pH. Cannot detect neutral species.

Osmolality is more exact measurement of urine solute concentration then specific gravity because specific gravity depends on the precise nature of the molecules presence in the urine.

Question 25

Describe viscometry and Poiseuille’s Law of Flow.

Answer 25

Viscosity arises because of a frictional force between adjacent layers of fluid as the slide past one another and can be defined as an internal resistance to flow for a liquid.

Poiseuille’s law: time required for a liquid to pass through a vertical tube is proportional to the viscosity of the liquid divided by its density

Measurement is reported as flow time relative to saline (measured in centipoise - cP). Affected by:

1. temperature (increase)
2. density (decrease)

Question 26

Describe viscometry and Poiseuille’s Law of Flow.

Answer 26

Viscosity arises because of a frictional force between adjacent layers of fluid as the slide past one another and can be defined as an internal resistance to flow for a liquid.

Poiseuille’s law: time required for a liquid to pass through a vertical tube is proportional to the viscosity of the liquid divided by its density

Measurement is reported as flow time relative to saline (measured in centipoise - cP). Affected by:

1. temperature (increase)
2. density (decrease)

Question 27

Describe a tube type (capillary viscometer).

Answer 27

Involves the passage of serum through a constricted glass or plastic tube. Measure the time for fluid to flow a predefined distance along the tube.

Question 28

Describe falling-body viscometers.

Answer 28

Examine the time for a sphere to fall a defined distance. Higher viscosity fluids cause the spheres to fall more slowly.

Question 29

Describe rotational viscometers.

Answer 29

Serum is placed in a narrow space between the spinning surface and the fixed surface. The more viscous the fluid, the more difficult it is to spin. Measure the speed of rotation and you calibrate against reference sample.

Question 30

List the clinical applications of viscometry.

Answer 30

1. Hyperviscosity syndrome:

Normal relative serum viscosity ranges from 1.4-1.8 cP.
Symptoms are usually not seen at viscosities greater than 5 units. Can cause vascular stasis and resultant hypoperfusion. Symptoms may include:
- mental disturbances due to increase viscosity of the blood and decreased cerebral flow
- visual disturbances due to dilation of retinal veins and retinal hemorrhages
- mucosal bleeding may occur from paraproteins, interfering with platelet function.

2. Hyperviscosity syndrome:

Commonly caused by plasma cell dyscrasia due to the large size of excess paraprotein (Waldenstrom macroglobulinemia, multiple myeloma, polycythemia, thrombocytosis, lukostasis).

Treatment is plasmapheresis for initial management and stabilization.

Question 31

Describe Stable Isotope Techniques.

Answer 31

Carbon-13:
- non-reactive, relatively abundant isotope of carbon
- suitable for paediatric and pregnant populations
C13 is now more affordable, incorporate into various substrates
- measurement of relative value (C12) vs absolute value

examples: urea breath test, lactose tolerance test

Question 32

Describe the urea breath test.

Answer 32

Urea is the carbon source for H pylori. Cleaved by urease to produce CO2. CO2 is then measured via scintillation or mass spectrometry. Non-invasive. (feed urea to the patient)

Question 33

Describe Isotope-Dilution Mass Spectrometry (IDMS).

Answer 33

Addition of a known amount of an enriched isotope of the element of interest to the sample.

Addition is made prior to the sample preparation during which the spiked addition is equilibrated to the sample.

Ratio of signal to isotope is calculated for both samples and calibrators and concentration can be determined using a curve.

Retention time drift, quenching and other related matrices effects do not present an interference with IDMS

Technique is considered a definitive method and is well suited and established for the certification of CRMs.