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Clinical Chemistry 1 > Lab Study > Flashcards

Lab Study Flashcards

1
Q

What must be done prior to running and reporting patient results?

A

Quality control results must be assessed.

If not done, results are considered invalid and patient results are not reported.

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2
Q

What is accuracy?

A

Accuracy refers to the closeness to the true value. Think hitting the ‘bull’s eye’.

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3
Q

What type of error can be detected using the mean?

A

Systematic errors that affect accuracy.

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4
Q

What is precision?

A

Precision refers to the agreement of test results to be reproducible. A deviation from precision can detect a random error.

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5
Q

What can you use to detect imprecision?

A

Standard deviation and coefficient of variation.

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6
Q

What are possible sources of systematic errors in the chemistry lab?

A
  1. Changes in reagent lot.
  2. Calibration issue (change in calibration or incorrect calibrators used).
  3. Deteriorating reagents.
  4. Deteriorating instrumentation (light source, pipettor alignment, etc.)
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7
Q

What are some sources of random errors in the lab?

A
  1. Bubbles (reagent, reagent lines, QC, etc.)
  2. Instrument instability (reagent dispensing error, light fluctuations, etc.)
  3. Temperature variation
  4. Human error (pipetting, improper mixing, etc.)
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8
Q

What are some corrective actions or things you can do to check for systematic errors?

A
  1. Check expiration dates (reagents/calibrators). Replace as req’d.
  2. Preventative maintenance. Investigate instrumentation. Calls service.
  3. Re-calibrate the assay.
  4. Ensure you are using the correct lot numbers of calibrators/reagents.
  5. Use correct charts w/ lot numbers.
  6. If no charts, may need to do a crossover study.
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9
Q

What is a crossover study / how is it done?

A

Crossover study is performed before the current lot of quality control (QC) material expires, or before the lab runs out of QC materials. The new lot is run in parallel with the current lot in order to establish new control limits.

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10
Q

What are some corrective actions / things to check for random errors?

A
  1. Check reagents and QC material for bubbles or that it has been properly mixed then re-run QC.
  2. Perform regular preventative maintenance on all instrumentation to avoid errors. Check if bubbles in lines or issues with pipettor dispensing.
  3. Follow SOPs.
  4. Ensure competency prior to performing technique.
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11
Q

What are quality control material in a chemistry lab?

A

QC material are stabilized specimens with a known range of results. They are made with the same matrix as the samples and are treated and tested like patient samples. Based on results obtained from QC material, a run may be accepted or rejected.

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12
Q

What is the control range? What percentage of all data points should fall within it?

A

Control Range is the range of acceptability, it is +/- 2SD (two std. dev.)

95% of all data points should fall within this range.

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13
Q

What are reference ranges (also called reference intervals)? How are these ranges determined?

A

Reference ranges are range of results that would be considered normal. \
Samples from a minimum of 120 “healthy/normal” patients that span a variety of ages, sexes, and ethnicities. (Child/adult though are different). Must be representative of the population the lab serves.

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14
Q

What is ‘inclusion’ criteria?

A

Inclusion criteria says that the people in the sample for the reference range should represent the population the lab serves. It identifies why these individuals should be in the study. These individuals have to complete a confidential questionnaire and sign a consent form.

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15
Q

What is exclusion criteira?

A

Exclusion criteria identifies those individuals that are considered outside the “healthy” population for that particular analyte due to a disease or disorder. They are not in the study for reference range.

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16
Q

What else is considered in determining reference ranges?

A

Pre-analytical variables are considered, i.e. if fasting is required for fasting glucose, hemolysis, lipemia, etc.

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17
Q

What is transference?

A

Transference is the term where labs use a reference range from made somewhere else with a similar instrument, similar lab, manufacturer/vendor and then validate using a method of comparison study or validate with a smaller population size such as 40.

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18
Q

What limits if transference can be done or not by a lab to determine their reference ranges?

A

It can only be done if an acceptable reference range already exists for the instrument or lab method to be employed.

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19
Q

If transference is being used, what is the acceptance criteria? What happens if it is not met?

A

If using transference, no more than 10% of the values obtained can be outside of the manufacturer’s reference range. E.g. if 40 samples, no more than 4 can be outside manufacturer’s range.

If not met, than a larger population needs to be sampled, 120 people, and the reference range re -calculated. If not met again, a still larger population sample is required.

If met, then transference can occur.

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20
Q

What is the difference between reference range and reportable range?

A

Reportable range represents the ability of an instrument to provided reliable analysis within a certain range. Higher or lower values than the range report as upper limit as instrument is unable to determine true value.

Reportable ranges are much broader than reference ranges. Typically the results below and above those limits are beyond healthy and are so extreme/life threatening and so are not necessary to actually determine.

The reference range helps determine what is normal.

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21
Q

How would you determine the reference range for a normal gaussian distribution of data? What if the data did not follow the normal gaussian distribution?

A

Reference range for a normal gaussian distribution of data would be = Mean +/- 2SD.

If the data was skewed this does not apply or you determining reference range for therapeutic drugs then the reference range is based on toxicity values or minimum effective concentrations.

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22
Q

What is method validation and name some procedures used?

A

Method validation is the process of determining if an analytical procedure is consistently reliable in producing quality lab results. Includes determining accuracy, precision, medical decision limits, diagnostic sensitivity, diagnostic specificity, and reportable range.

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23
Q

What are medical decision limits?

A

Medical decision limits are values for analytes that represent specific diseases.

Qualitative medical decision limits - presence if an analyte if there or not correlates to disease state or absence of disease state.
Quantitative medical decision limits - are cutoff concentrations that if above or below correlate to a specific disease state.

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24
Q

Good medical decision limits will have low ________ and low ______ results.

A

False-positive

false-negative

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25
Q

What is the reportable range (or analytical range, AMR, or dynamic range)?

A

Reportable range is the range of specimen concentration (lowest to highest) which the method may be used without dilution.

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26
Q

How do you determine reportable range?

A
  1. Use elevated patient samples, spiked patient pool, or commercially available linearity materials. Must have same sample matrix as patient samples.
  2. Serially dilute to at least 5 to 7x’s to equally cover reportable range.
  3. Do testing in duplicate.
  4. Run from lowest to highest to avoid any potential carryover.
  5. Linear regression analysis is performed and compared to the manufacturer data (y = mx + b).
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27
Q

In the linear regression analysis for checking reportable range, what are the potential results?

A
  1. No error, lines overlap or have similar slope and y-intercept.
  2. Constant error, the slope is the same but the y-intercept is different (b is off).
  3. Proportional error, change in slope of line (difference in m).
  4. Combination of both of the above.
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28
Q

Between the manufacturer’s line and your analysis for linear regression what statistical value can be used to evaluate how well the two lines match?

A

Calculate Correlation of determination, R^2, value from 0 to 1. In chemistry, we aim for R^2 > 0.98.

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29
Q

What is the lower limit of detection (or analytical sensitivity)? How is it determined?

A

Lower limit of detection - lowest amount of analyte that can be accurately detected by the method. Removes low-end interferences, also known as noise from being read as false-positives.

A ZERO CALIBRATOR is run 20 times. Std. dev and mean is calc’d. Lower limit of detection is 3 x Std. Dev above the mean of the zero calibrator results.

30
Q

What is the limit of quantification (LoQ)?

A

LoQ represents the lowest concentration that can be reliably quantified and reported.

31
Q

What is a spectrophotometer?

A

An instrument that can measure the amount of monochromatic light (radiant energy of one wavelength) that can be absorbed or transmitted through a solution.

Each component of the spec. plays a role in the manipulation and measurement of light so the concentration can be determined.

32
Q

How do you obtained the optimum wavelength of a coloured solution and why is that important?

A

A spectral absorbance curve is made to determine the optimal absorbance.

It is important because then a standard (calibration ) curve can be made using the optimal wavelength with a linear relationship. From there, the concentration of the unknowns can be determined.

33
Q

What components does a basic spectrophotometer consist of?

A
  1. Light source (radiant w/consistent intensity, tungsten lamps for visible light)
  2. Monochromator, collimator to prism or grating to wavelength selector.
  3. Sample Holder, where you place cuvette with solution inside it. Typically plastic, 1cm wide light path.
  4. Photo-detectors - converts transmitted light into an electrical signal, e.g. photocell, phototube, photomultiplier tube, photodiode array, etc.
  5. Digital Display - to show absorbance or transmittance readings. (Or sent to computer w/software).
34
Q

What QC issues could occur with the use of the cuvette?

A
  1. Watch for scratched cuvettes or dirty ones w/ fingerprints, dust, and other debris. Wipe with Kim Wipe prior to inserting into sample holder. MUST CLEAN, FREE of DEGECTS.
  2. ALIGN. Align orientation marking (arrow, triangle, or a clear versus opaque side) so the cuvette is correctly aligned in the light path.
35
Q

How do you create a spectral absorbance curve to find the optimal wavelength?

A
  1. Take readings starting at ~400nm, every 20 nm intervals until 700 nm. Ensure to reset 0 absorbance between each wavelength change.
  2. Locate evident peak and fine-tune optimal wavelength by measuring at 5 nm internals on either side of that peak.
36
Q

How can you use a table of wavelength ranges and colour of light absorbed versus colour of light transmitted to aid where you expect to find the absorbance peak?

A

If you solution was blue that is the colour of transmittance (light reflected or transmitted) so then the light absorbed is orange and the wave length range is 600 to 650 nm.

37
Q

How is the standard (calibration) curve created after the optimal absorbance wavelength is found? How to calc unknowns from this curve?

A

Measure known concentrations of solution ranging from low to high - referred to as standards. The more standards used the more accurate the standard curve. Beer’s law says we should get a linear relationship of absorbance varying with concentration. From the linear relationship equation determined we can calculate concentration of the patient unknowns.

38
Q

What are the two methods of glucose measurements?

A

Glucose oxidase and hexokinase methods.

39
Q

In the glucose oxidase method, how can glucose concentration be determined from this 2-step reaction?

A

The 2step reaction produces an intense brown colour measured at 425 to 275 nm. The absorbance is proportional to the original glucose concentration in the patient serum.

40
Q

Which method is considered more accurate? Why?

A

The hexokinase is considered more accurate. This makes it the recommended reference method of glucose quantification in the lab.

Glucose-6-Phosphate dehydrogenase is highly specific and not as susceptible to interference from uric acid or ascorbic acid.

41
Q

What is measured in the hexokinase method?

A

NADH (or NADPH) production is measured at 340 nm. The rate of production is proportional to the amount of glucose in the patient’s serum.

42
Q

What can the hexokinase method also measure?

A

It can also measure glucose levels in urine, cerebrospinal fluid, and serous fluids.

43
Q

What can cause falsely decreased results in the hexokinase method?

A

Falsely decreased results can occur with gross hemolysis or extremely high bilirubin.

44
Q

The glucose oxidase and hexokinase are the only method utilized in the lab to measure glucose, what is important to know about the method your lab is using when it comes to accurate and precise results?

A

You need to know the method and understand the interfering substances so you can ensure accurate and precise results.

45
Q

How do you find concentration using the point correlation method (not sure of term here)?

A

If the concentration of the patient blood glucose value is anticipated to be similar to the concentration of a standard, you can calculate the concentration using Cu = (Au/As) x Cs. If the comparison concentration and absorbance are not close to the expected value than it could result in a less precise result.

46
Q

What does PCR mimic?

A

The “in vivo” process of nucleic acid replication to generate enough material for analysis within the lab.

47
Q

What is all included in a PCR master mix?

A
  1. Distilled DNAase/RNAse free water - diluent.
  2. Buffer - for optimal pH
  3. Magnesium, usually in buffer as a co-factor for polymerase activity.
  4. dNTPs as the nucleotide building blocks (A, T, G, and C).
  5. Taq polymerase as the enzyme to synthesize PCR product.
  6. Nucleic acid sample for the template.
48
Q

What could contaminate our PCR reaction? How is this mitigated?

A
  1. Avoid introduction of amplicons (other?) - could cause false positive results
  2. Contaminating nucleotide sequences that could mask or interfere with testing and consume ingredients from master mix.

Molecular labs have a one way work flow to mitigate contaminates affecting results.

49
Q

How does the clean to dirty workflow work?

A

Areas deemed “clean” (free of template) and “dirty” (area w/ amplicons). Master mix is created in the clean space, template added in a different area, then that is transferred to the “dirty” area where the thermocycler is located. Once you are in the dirty area you cannot go back to the clean area.

50
Q

What are other precautions taken to avoid contamination?

A
  1. CLEAN. Areas are frequently cleaned with diluted bleach, RNAse/DNAse Away and/or 70% ethanol solution.
  2. PPE. Worn to protect yourself and to protect your product from contamination and RNAse from you.
51
Q

What are the three stages each PCR cycle consists of? How much product is created each cycle?

A
  1. Denaturation
  2. Annealing
  3. Extension

Each cycle 2^N product is created where N is the number of PCR cycles.

52
Q

What are the temperatures for each stage of each cycle and what temp does it run at?

A
  1. Denature - 90-96C, breaks hydrogen bonds between nucleotides causing the strands of the double helix to come apart.
  2. Anneal - primers attach to the template strand near the desired sequence and act as an attachment site for complementary nucleotides to be added. Temp is 5C below the melting temperature (1/2 ds and 1/2 ss), ~50-70C.
  3. Extend - 68-75C. For optimal activity of the Taq polymerase enzyme to synthesize PCR product. Attaches dNTPs starting and extending from the primers using the template strand to create a complementary sequence.
53
Q

What happens if the denature temp is too high?

A

If temp is too high it will cause damage to the phosphodiester bonds of the “backbone”. GC strands with 3H bonds vs the 2H bonds of At may require a slightly higher temperature or more time during the denature step.

54
Q

What is Gel Electrophoresis used to do for DNA?

A

A technique that separates nucleic acid fragments according to their size based on their relative charge.

55
Q

How does gel electrophoresis work?

A
  1. DNA is introduced into wells at one end of the a gel closest to the neg electrode and an electrical current is applied.
  2. A dye is applied to visualize the bands (and also sometimes UV light) so interpretation can be made as nucleic acids are not visible.
  3. Smaller fragments of DNA will travel further and faster through the gel and the larger fragments will remain closer to the well.
  4. Interpretations are based on comparison of the banding pattern of the DNA compared to a molecular weight ladder and controls.
56
Q

What is a molecular weight ladder?

A

Fragments of known sizes, so you can interpret the number of base pairs in a band.

57
Q

What is the charge of each nucleotide? How does this affect the gel electrophoresis?

A

One negative charge per nucleotide.

Migration in the gel is inversely related to the size of the DNA sequence. The smaller the sequence the further and faster it will travel through the gel. If a sequence is large it will not travel far from the point of introduction (i.e. the well).

58
Q

Where are the wells positioned?

A

Close to the negative electrode.

59
Q

How does the DNA migrate?

A

DNA migrates from the negative electrode towards the positive electrode within the agarose gel.

60
Q

What kind of gel is used for the gel electrophoresis experiment and why?

A

Agarose gel. It is a neutral polymer derived from seaweed. It is porous so DNA can easily migrate through.

The concentration used in the procedure depends on the size of the desired DNA bands.

61
Q

What determines the concentration of agarose desired for gel electrophoresis?

A

The concentration desired depends on the desired size of DNA bands. The higher the concentration of agarose, the smaller the size of the nucleic acid sequences needing to be separated.

The gel is also permeable to dyes.

62
Q

What is the purpose of the buffer placed around the gel? What should the buffer not affect?

A
  1. The gel is submersed in a buffer which maintains the pH and allows for the passage of the electrical current.
  2. Buffer should not denature the DNA or cause increased conductivity which would disrupt the integrity of the agarose gel.
63
Q

What are common buffers used to submerse the gel into?

A

Tris borate EDTA or

Tris acetate EDTA.

64
Q

What are the purpose of loading dyes and when are they added and where?

A

Loading dyes make the DNA visible.
It also weights the sample down so it does not float off into the buffer and migrates at approximately the same rate as the smallest fragment in your sample so you know your electrophoresis apparatus is set-up appropriately.

Added to the well at set-up (after DNA is added) Check procedure.

65
Q

What loading dye was noted in our lab notes?

A

Bromophenol blue with glycerol.

66
Q

How much time do you allow to elapse for the gel electrophoresis when you turn the current on?

A

Enough time to allow the nucleic acids to migrate through 1/2 to 3/4 of the length of the gel.

67
Q

After electrophoresis what dye is used to illuminate the DNA under UV light? Wavelength of UV light?

A

Ethidium bromide.
UV light at 300 nm.

Ethidium bromide can be added to a solution of diluted buffer so the whole gel is submersed following electrophoresis or some labs may impregnate the dye directly in the gel to better control the use of this potent mutagen.

68
Q

What wavelength does ethidium bromide emit and its colour?

A

Ethidium bromide emits light at 590 nm and appears orange.

69
Q

What other analysis can gel electrophoresis be used for?

A
  1. Detection technique following treatment with restriction enzymes such as Restriction Fragment Length Polymorphism analysis.
  2. Looking for the absence or presence of a target sequences following PCR.
70
Q

What percentage falls within +/- 1SD, +/- 2SD and +/-3D? (for your info)?

A

From slide 28:
+/- 1SD 68.2% (31.8% outside)
+/- 2SD 95.5% ( so 4.5% outside)
+/- 3SD 99.7% ( 0.3% outside)

Clinical Chemistry 1 (11 decks)

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