Lab Study 2 Flashcards
What does the biuret reagent contain?
The Biuret reagent contains:
- Copper sulfate pentahydrate - source of cupric ions (Cu2+)
- Sodium hydroxide - creates an alkaline environment
- Sodium potassium tartrate - forms complex with cupric ions to prevent their precipitation within the alkaline environment
- Potassium iodide - antioxidant
What colour will the proteins change to with the biuret reagent? Why?
Light pink (small peptide chains) or violet (presence of many peptide bonds).
In an alkaline environment, the copper atoms will form a complex with the nitrogen of peptide bonds in polypeptides producing a color change from blue to a light pink (presence of small peptide chains) or violet (presence of many peptide bonds) as the chelate forms. No color change indicates the absence of peptide bonds, and therefore, is negative for proteins.
What is the reference range for total protein? What does total protein consist of?
The reference range for total protein is 60-80 g/L. Total protein consists of albumin and globulins. Each protein fraction can be quantified using other methods.
For pre-analytical considerations, what problem samples should be avoided? What do you do if you can’t avoid these issues?
- a) Lipemic, icteric, and hemolyzed samples should be avoided as it interferes with the test
b) Free hemoglobin causes elevated results - If unavoidable can correct with a serum blank.
For pre-analytical consideration, how are ambulatory patients results affected? Why?
- Protein values are approximately 5 g/L higher for ambulatory patients (slight hemoconcentration) than patients that are supine or recumbent
- Reduction in serum albumin and fluid shift toward the extracellular compartments
How does pregnancy affect total protein counts?
Lower total protein results are seen in pregnancy.
What is the preferred sample type? (i.e. plasma or serum).
Serum is the preferred sample type. Plasma can be used but will give a slightly higher value due to the fibrinogen content.
How much sample is used in Serum Protein Electrophoresis?
2 - 10 uL applied to a support medium.
What support medium materials are used in serum protein electrophoresis?
The support medium, a neutral polymer such as agarose, cellulose acetate, or polyacrylamide provides structural support for the sample to travel through.
After ~5 mins of allowing the sample to diffuse into the support medium, the support medium is put into an electrophoresis chamber containing buffer, what does the buffer do? What pH is typically used?
The buffer maintains the pH and ionic strength within the electrophoresis environment. Generally, a buffer with a pH of 8.6 is used for separation of serum proteins. It is important that the buffer maintain contact with the support medium to maintain consistent and constant electrical flow.
Where are the serum samples placed? Where do the serum proteins migrate to?
There are two electrodes, the cathode (negative electrode) and the anode (positive electrode). Serum samples are applied close to the cathode end of the support medium and will migrate towards the anode as many serum proteins carry a net negative charge in a buffer at pH 8.6. So, the current travels between the electrodes through the buffer and medium separating the proteins from the sample.
What is the next two steps after electrophoresis for serum proteins?
- Once the appropriate amount of time has passed, the support medium is removed and placed in a fixative (usually an acidic solution) or dried to prevent further migration and prevent loss of sample from the medium.
- The support medium is then stained in order to visualize the banding pattern. Excess stain is washed away to ensure it does not interfere with interpretation.
What is the order of the bands in terms of type of proteins?
The dark band closest to the anode is albumin (dark because it is the most abundant serum protein), then α1, then α2, then β (actually comprised of a β1 and β2 band), and finally γ (more faint and wider in normal serum) located closest to the cathode.
How do you know which protein is in the greatest quantity in your sample without using a densitometer?
The amount of dye taken up by the band is proportional to the amount of sample present in that area.
What can be used to estimate the quantities of each protein in the sample?
The stained support medium can be read using a densitometer, an instrument that accurately quantifies the amount of protein fraction present using optical density (remember Beer’s Law!). The wider and darker the band, the wider and higher the peak produced in the densitometric pattern. The area under each peak represents the proportion of that protein fraction in the sample.
How do you interpret the banding or densitometric pattern?
We must first understand what the expected normal values are in regards to the serum fractions as part of total protein values.
See table in lab. Protein Frac. Ref Range(SI) %of total Pro. Albumin 35-50 g/L 53-65% α1 1-3 g/L 2.5-5% α2 6-10 g/L 7-13% β 7-11 g/L 8-14% γ 8-16 g/L 12-22%
What do increased or decreased values potentially mean for each protein fraction, albumin, alpha1, alpha2, beta, and gamma?
Protein Fraction / Increased Values Seen In / Decreased Values Seen In:
Albumin: I: Dehydration
D: Malnutrition,
Inflammation, Liver and
Kidney Disease
α1 I: Infection and inflammation
D: Liver Disease
α2 I: Inflammation, infection and
kidney disease
D: Liver Disease
β I: Iron deficiency anemia
D: Poor nutrition
γ I: Infection, inflammation, liver
disease, “M” spike with Multiple
Myeloma
D: Hypogamma-globulinemia
What occurs with alpha2 and beta fractions when samples are hemolyzed?
It will sometimes just appear as an increased beta band.
What do you see in the cases of cirrhosis?
A beta-gamma bridge.
What do you see if plasma is used instead of serum?
A narrow band will appear between beta and gamma fractions.
What variables can influence the success of your electrophoresis run?
Human competence, pH, buffer strength, gel integrity, voltage, time, etc.
What affect can too much heat cause with electrophoresis of proteins?
Wick flow:
Heat causes evaporation of buffer, drawing the support medium in from the edges causing an uneven pattern
What is ‘Electroendosmosis’ and how can it affect the results?
With basic buffers, proteins take on a negative charge and migrate towards the positive electrode (anode). The buffer contains positive ions that form a positive ion cloud which migrates to the cathode. This movement in opposite directions slows migration, or causes the large or less charged proteins to migrate in the opposite direction
What are some simple problems that can cause no migration?
No power,
No buffer, and
No sample.
What are some reasons that could cause indistinct bands?
- voltage too high,
- incorrect migration time (long patterns = time too long;
- bands not separated = too short), or
- low ionic strength buffer
What could cause holes in the pattern?
Air bubbles in support medium or in sample.
What are immunoassays?
Immunoassays consist of a wide range of methods used to identify and quantify analytes by manipulating the known characteristics of antigen and antibody interactions.
What are antibodies?
Antibodies are glycoprotein molecules that bind “lock and key” to antigens.
In body –> Function to remove “non-self” antigens (such as those on bacterial surfaces) through the processes of neutralization and/or opsonization.
What is the specific and unique area of the antigen that the antibody binds to called?
Epitope.
How can be use antigen or antibodies for analysis?
If the antigen or antibody used in the method is known, then we can determine the presence, absence, or quantity of the other.
Known antigen investigate unknown antibody and vice versa.
What type of substances can be investigated for using antibody/antigen reactions?
Presence of pathogens Hormones, Drugs Current infection Long term immunity
What are the benefits of antigen-antibody reactions?
Quick
Accurate
Precise laboratory results.
What is the difference between performing a heterogeneous and homogeneous immunoassay?
Heterogenous immunoassays require a physical SEPARATION step of the Ag:Ab complex from other sample components.
Homogeneous immunoassays DO NOT.
What is a “competitive” immunoassay?
Competitive Immunoassay:
A analyte in the patient sample competes with a labelled analyte in the reagent.
How are the amounts of labelled antigen related to the patient analyte?
The amount of signal produced from the labelled Ag:Ab complex is inversely proportional to the amount of analyte present in the patient sample.
What is a noncompetitive immunoassay?
Noncompetitive Immunoassay:
No competition between the analyte and reagent.
What are different types of noncompetitive immunoassays?
- Direct (using one labelled analyte in reagent).
- Indirect. E.g. Two antibodies can be used, one unlabeled and fixed to the well and the other labeled that attaches to the antigen present in the patient sample, forming a sandwich.
Note: Confirm understanding re #2.
How is the amount of signal produced from the labelled Ag:Ab complex related to the amount of analyte present in the patient’s sample in noncompetitive immunoassays?
Directly proportional.
What are heterophile antibodies and how can it interfere with the results?
Heterophile antibodies are antibodies in patient serum that can react with reagent antibodies causing both false positive and false negative results.
What is the hook effect and how can this impact immunoassays?
The hook effect (aka prozone effect) occurs when there is an excess of antigen in the patient sample causing a saturation of reagent antibody. This causes results to be lowered or even false negative.
How does one counteract the ‘hook effect’ on immunoassays?
As there is an excess of antigen in the patient sample, the sample must be diluted and reanalyzed.
What are some different immunoassay methods (list but don’t give details yet)?
- Radioimmunoassay (RIA)
- Enzyme Immunoassay (EIA) or Enzyme Linked Immunosorbent Assay (ELISA)
- Fluoroimmunoassay (FIA)
- Chemiluminescence Immunoassay (CLIA)
- Immunochromatographic Assay (ICA) (aka as lateral flow immunoassay).
What is a radioimmunoassay (RIA)?
Utilizes radioisotopes (I-235) to label the reagent antibody or antigen. As a competitive assay, the analyte in the patient sample competes with labeled reagent analyte. Unbound reagent and patient sample is washed away. The radioactive signal is inversely related to the amount of analyte. Highly sensitive. Not as common anymore due to health risk associated with radioisotopes
What is a Enzyme Immunoassay (EIA) or Enzyme-Linked Immunosorbent Assay (ELISA)?
Utilizes enzymes as the label such as horseradish peroxidase (HRP) and alkaline phosphatase (AP). Following incubation, unbound analyte is washed away. Detection occurs through a colour change following the addition of a substrate and chromagen in order to visualize the antigen:antibody complex. There are many different methods of ELISA (direct, indirect, and sandwich).
What is a Fluoroimmunoassay (FIA)?
Utilizes a fluorophore label. Following incubation, the antigen:antibody complex is isolated. The fluorescent signal is measured using a fluorometer.
What is Chemiluminescence Immunoassay (CLIA)?
Chemiluminescent labels (luminol, isoluminol, etc.) produce a signal when antigen:antibody complex forms. Chemiluminescence is achieved through a chemical reaction which can be quantified.
What is Immunochromatographic Assay (ICA) (aka as lateral flow immunoassay)?
Rapid testing method. Sample analyte travels through porous material until it meets immobilized capture antibodies. As complex accumulates a visible product is produced thanks to colloidal gold nanoparticles in the membrane.
What are the benefits of automated immunoassay methods?
Automated immunoassay methods allow for
- greater lab efficiency by increasing the throughput of samples,
- quicker turn-around-time, and
- time for the technologist to perform other tasks in the laboratory while an assay is being performed.
What does the Cobas e 411 do?
This analyzer uses electrochemiluminescence (ECL) technology for immunoassay analysis. It comes as a rack or disk sample handling system.
What feedback controls the synthesis and secretion of TSH? What gland secretes it?
- Thyroid stimulating hormone (TSH) is synthesized and secreted by the anterior pituitary gland in response to circulating levels of free T3 (triiodothyronine) and free T4 (thyroxine).
- It is also under direct control of the thyrotropin-releasing hormone (TRH) secreted by the hypothalamus.
What is the first step in assessing thyroid disorders and therapies?
Determination of TSH levels is the first step in assessing thyroid disorders and therapies.
How do changes in free T3 and free T4 affect TSH?
Slight changes in circulating free T3 and free T4 levels result in opposite changes in TSH level.
What is done in the lab when there is abnormal TSH?
Abnormal TSH will reflex Free T4/ Free T3 testing as appropriate.
- Free T4 will be performed when TSH < 0.40 mU/L and ≥ 4.3 mU/L
- Free T3 will be performed when TSH < 0.09 mU/L
What are the adult reference ranges for TSH and Free T4?
TSH adult reference range: 0.4-4.2 mU/L
Free T4 adult reference range: 9.7-25.7 pmol/L
Compare the levels of TSH and FT4 for:\
a) Normal Thyroid Function
b) Hyperthyroidism
c) Primary Hypothyroidism
d) Secondary Hypothyroidism
a) Normal Thyroid Function:
Normal TSH & FT4.
b) Hyperthyroidism:
Low TSH, High FT4
c) Primary Hypothyroidism:
High TSH, Low FT4
d) Secondary Hypothyroidism:
Low TSH, Low FT4By what principles does the Cobas e411 perform analysis on TSH and FT4?
The Cobas e 411 utilizes the sandwich immunoassay principle for TSH analysis, but a competitive immunoassay for FT4 analysis.
What is the purpose of HCG in pregnancy? What produces it and when?
Human chorionic gonadotropin (HCG) is produced by the placenta and serves to maintain the corpus luteum during early pregnancy.
How early in pregnancy can HCG be detected in plasma? How does it increase in time?
In pregnancy, plasma contains mainly intact HCG which can be detected as early as 1 week after conception, and will double every 2 to 3 days for the first 6 weeks.
Besides the corpus leteum in pregnancy what other tissues can produce hCG?
HCG can also be produced by trophoblastic tumours, germ cell tumours, and some non-trophoblastic tumours.
What should the analysis include if hCG is being measured to monitor disease?
If HCG is being monitored for trophoblastic disease, then analysis must include measurement of the whole molecule as well as the free beta subunit.
What is the non-pregnant female and male reference range for hCG?
HCG male reference range: < 5 IU/L
What level of hCG does a female have in the first week of pregnancy? second week?
0 - 1 week: 0-15 IU/L
1 - 2 weeks: 40-400 IU/L
What is the best time to take a urine pregnancy test?
First morning urine should be used to determine the presence of human chorionic gonadotropin (hCG), the hormone produced by the developing placenta shortly after fertilization.
What is the procedure for the ICON 25 hCG test?
- Bring the pouch to room temperature.
- Open the pouch and remove the testing device, placing it on a clean and level surface.
- Mix sample thoroughly
- Aspirate some sample using a transfer pipet.
- Hold the transfer pipet vertically and drop three full drops of urine to the sample well (S). Avoid introducing air bubbles.
- Wait three minutes.
- Read and record results.
a) A red line must form at the control (C) region in order for results to be valid
b) A red line in the test (T) region is interpreted as positive
c) The absence of a red line in the test (T) region is interpreted as negative
What are the most commonly encountered methods for high resolution separation of chromatography?
Gas chromatography (GC) and high-pressure liquid chromatography (HPLC), as well as mass spectrometry which is often coupled with these instruments as a detector.
Does chromatography instruments provide qualitative or quantitative analysis?
Chromatography instruments provide for accurate and precise qualitative and quantitative analysis.
What is gas chromatography?
Gas chromatography (GC) separates mixtures containing compounds that are volatile or compounds that can be made volatile.
What state must the sample be in when entering a gas chromatography instrument?
The sample must be in a gaseous state prior to entering the separation column. Therefore, it can either be injected through a septum as a gas or the temperature of the injection port must be higher than the boiling point of the compounds in order to vaporize the liquid sample upon injection.
What is the mobile phase and what does it do in gas chromatography?
The vaporized sample is then carried through the column by the mobile phase, a chemically inert carrier gas such as nitrogen, helium, hydrogen or argon.
What are columns made of in a gas chromatography instrument?
Columns can be made of glass, fused silica, Teflon, or stainless steel and packed with diatomaceous earth, porous polymer, or glass beads coated with a nonvolatile liquid.
How does the type of material the column is made of affect what type of gas chromatography can be done?
Depending on what the column is made of the stationary phase can be either solid (Gas-Solid Chromatography - GSC) or liquid (Gas-Liquid Chromatography - GLC).
What are the typical column widths and lengths? How are they fitted into the oven?
Columns are 3-6 mm wide and can be 2-60 meters long. For this reason, the columns are coiled so they can fit in the oven.
What is the purpose of the oven in gas chromatography?
The oven ensures proper column temperature to best optimize compound resolution.
How do various components of the samples move through the column? End result?
- Part of the sample will remain gaseous as it passes through the column and part of it will interact with the solid or liquid stationary phase.
- Volatile compounds that are mainly gas will move freely in the column and pass through quickly.
- If the interaction with the stationary phase is strong, compounds will be adsorbed temporarily and exit the column more slowly.
- Eventually, compounds will pass through a detector that produces an electrical signal.
What are the different type of detectors of gas chromatography?
Detectors for GC include: thermal conductivity, flame ionization, mass spectrometry, etc.
What does the gas chromatography instrument use to identify and measure concentrations of compounds?
The chromatogram can be used to identify the compounds according to their retention time and concentrations can be determined by calculating the area under the peak.
What is the typical clinical application of the gas chromatography instrument?
This instrumentation is seen in the toxicology department to test drug metabolites from patient samples.
What is HPLC?
- HPLC: In high-pressure liquid chromatography (HPLC) separation can occur faster with the use of pressure. 2. Essentially, a pump forces solvents (liquid mobile phase such as methanol, ethyl acetate, acetonitrile, etc ) and the sample through an analytical column (stationary phase).
- This is what gives H”P”LC its name, with the “P” representing pressure or performance.
What are the two different phases of HPLC?
- The sample is introduced in the path of the solvent using loop injectors.
- The solvent and sample is then forced into the column.
There are two different phases of HPLC.
What is the difference between normal-phased and reversed-phase HPLC?
- Normal-phased HPLC has a non-polar mobile phase and a polar stationary phase.
- Reversed-phase HPLC is the opposite with a polar mobile phase and a non-polar stationary phase, so non-polar molecules are retained longer while polar molecules move through the column with the mobile phase.
What are the typical materials of columns in HPLC?
Most columns are stainless steel columns packed with silica gel (solid), or stainless steel columns packed with solvent coated silica gel (liquid).
What temperature are columns ran at in HPLC?
Most columns are run at ambient temperatures, but some can be housed in an oven to enhance resolution.
Comparatively are long are the columns used in HPLC compared to those used in gas chromatography?
The columns are significantly shorter than those used in gas chromatography.
What are some type of detectors used as the eluate leaves the column in HPLC?
A detector monitors the eluate as it leaves the column. Some examples of detectors used in HPLC include: UV spectrophotometry, electrochemical detector, mass spectrometry, etc.
What is used to analyze results from a HPLC?
The signal allows for creation of a CHROMATOGRAM allowing for identification of compounds based on their retention time and determination of concentrations of those compounds based on peak analysis.
What are the typical clinical applications of HPLC?
This instrumentation is seen in the clinical laboratories testing for newborn screening, Vitamin D, drugs of abuse, hormones, immunosuppresant drugs, amino acids, etc.
Where is a mass spectrometer used in gas chromatography or HPLC?
A mass spectrometer can be used as the detector following separation of samples with gas chromatography or high-pressure liquid chromatography.
When these techniques are coupled they are referred to as GC/MS and LC/MS, respectively. There have even been some advances that have allowed for direct injection of samples into a mass spectrometer.
What happens to the compound or sample in a mass spectrometer?
The separated compounds or sample are volatilized and ionized to form charged fragments that are detected according to their mass-to-charge ratio (m/z).
What are the basic components of the mass spectrometer?
- Sample inlet
- Ionization source
- Mass analyzer
- Ion detector
What occurs at the sample inlet in a mass spectrometer?
- Sample is introduced into mass spectrometer directly from GC or HPLC. 2. For some advanced methods, sample introduction can occur directly
What are the two methods of ionization source?
Ionization Source Methods such as:
- Electron ionization - sample molecules are bombarded with high-energy electrons at 70 eV potential. Molecules are broken down into characteristic charged fragments based on their molecular structure.
- Electrospray ionization - ions are produced using an electrospray. High voltage is applied to the effluent to create aerosolized charged droplets.
What does the mass analyzer do?
Generation of electrical and magnetic fields that manipulate the charged molecules in order to accelerate, deflect, and finally sort them according to their mass to charge ration (m/z). Only the selected ions are able to pass through to the detector while others are deflected. This all occurs within a vacuum to allow ions to move freely without interference with other ions.
What does the ion detector do?
Detection occurs via an electron multiplier system that creates an electrical signal.
How are results represented often from a mass spectrometer?
Results from a mass spectrometer can be presented as a mass spectrum with intensity versus mass-to-charge ratio (m/z).
What does the mass spectrum mean and how do you interpret it?
The pattern created represents the distribution of ions in a sample. The ion in greatest abundance will appear as the tallest line.
What is a chromatogram and describe each graph axis?
A chromatogram is a visible display, usually a graph, that is created in real time as analytes are being detected. A basic chromatogram will have peaks representing different analytes,
- retention time on the x-axis, and
- the signal generated by the analyte on the y-axis.
The mass spectrum is used to produce a characteristic graph for each component separated by chromatography.
What is the definition of retention time?
Retention time is defined as the time interval between sample injection and the maximum of the peak.
What impacts retention time?
The retention time is unique to each specific analyte under the same operating conditions. Retention time can be impacted by variables such as flow rate, injection temperatures, oven temperature, column diameter and length, etc.
How are the analyte peaks analyzed? And how is it used to determine concentration?
Different analyte peaks are created at different retention times. Therefore, the peak generated can be analyzed to identify the particular analyte under certain operating conditions and the area under the peak can be calculated to determine the analyte’s concentration.
