Analytics Flashcards
1
Q
Protein purification advantage and disadvantage
A
Ad- fast growth rate, cheap, transform bacteria from plasmid dna
Dis- proteins fold incorrectly, lacks some post transitional bodies
2
Q
Protein purification
A
- Lyse bacteria cell walls without denaturing protein if Interest, freeze thawing, triton x non ionic detergent sonication
3
Q
Purify protein from extract lysate
A
Differential solubility, Affinity chromatography, Size exclusion chromatography, Ion exchange chromatography, Hydrophobic interaction chromatography, Isoelectric Focusing
Can require multiple rounds of purification
4
Q
Track protein in purification process
A
Track protein in purification process by western blotting - Primary antibody binds target protein & Secondary antibody with tag for detection allows visualisation
5
Q
Protein assay - Bradford assay / Bicinchoninic acid (BCA) assay
A
in alkaline solution - proteins reduce Cu2+ to Cu1+
Cu1+ complexes with BCA (Purple → Darker purple = more protein)
Include a range of known protein concentrations - Allows the construction of a standard curve
6
Q
Enrichment factor- protein purification
A
Yield (%) = (enzyme activity after purification step / enzyme activity in original sample) x 100
Enrichment factor = specific activity after purification step / specific activity in original sample
Aka purification factor
Dependent on - origin of the starting source.& efficiency of different steps.
7
Q
Differential solubility
A
Initial purification step
Polar water molecules interact with hydrophilic regions of protein - increases protein solubility - Oxygen in water more electronegative
Anything affecting protein charge, structure or water interaction affects solubility
8
Q
Ammonium sulphate precipitation - salting out with high salt concentration
A
Proteins fold - charged/ polar amino acids = hydrophilic protein surface
Uncharged hydrophobic amino acids hidden inside structure
Proteins are solubilised by hydrogen bonding with polar water molecules
Addition of high salt concentration = displacement of the water molecules & precipitation of the protein
Water binds with salt ions instead of proteins
Different proteins have different solubilities in aqueous solution
Ammonium sulphate - Highly water-soluble, cheap. No permanent denaturation
9
Q
Salt Removal and Buffer Exchange
A
Salt may not need to be removed prior to next purification step – gel filtration chromatography and hydrophobic interaction chromatography
Dialysis
Sample is placed in a bag with semi-permeable membrane - permeability based on target protein
Pores too small to allow passage of your protein - but big enough to allow passage of salt ions
Several changes of buffer eventually remove the salt from your sample
Gel filtration - pores separates sample components based on size
Load dissolved protein (and salt) onto column – flush sample through with buffer
Small salt ions enter the pores of resin, whilst large proteins pass straight through (carried in the buffer)
Diafiltration - Pressure-driven filtration membrane
Salt passes through membrane BUT Protein is retained in sample
New buffer can be added and protein can also be concentrated
10
Q
pH & protein solubility
A
Proteins have an overall charge - dictated by the presence of amino acid side chains that can gain or lose H+
overall protein charge changes with pH
Charged amino acids are hydrophilic – form hydrogen bonds with water, increasing protein solubility
Isoelectric point - pH where a protein has no net charge
least solubility due to lack of interaction with water molecules - precipitation
Heat denaturation
Heating = denature →exposes hydrophobic areas that bind each other, causing protein precipitation
Some proteins don’t unfold after heating- thermos table above 45 degrees
11
Q
Affinity resin/matrix composed of an affinity molecule
A
bound to a solid support
e.g. Sepharose beads
Affinity matrix specifically recognises protein of interest - Protein may have specific tag
Beads can then be centrifuged and washed, removing unbound extract components (batch purification)
Purified target protein can then be eluted from beads
Affinity resin can also be packed into a column (‘column purification) for larger scale purifications
Add cell extract, then several wash steps and then elute target protein
Wash steps with increasing NaCl concentrations
12
Q
His Tag purification
A
His-Tag purification
Bacterial expression vector for the production of a His-tagged protein
His-tags bind strongly to beads coated with nickel (Ni2+)
Often used for purification of proteins from bacteria
13
Q
Epitope tags
A
often used for protein detection, affinity tags for purification
Consideration - potential functional effect of attaching a large tag
N-terminal or C-terminal
Can also tag proteins with biotin – binds strongly to streptavidin
GFP tags (big ~30kDa) - allow visualisation of proteins (and immunoprecipitation) using ‘GFP-TRAP’ (Chromotek)
Some tags can be removed after purification (e.g. enterokinase cuts after DDDDK in FLAG tag)
14
Q
Gel filtration chromatography
A
separates proteins based on size
Add cell extract and allow to pass through column with buffer (mobile phase)
Collect multiple fractions over time – increasing volumes of buffer
Elution volume = the volume of buffer at which a particular protein exits the column
Monitor protein elution with UV absorbanc
15
Q
Bigger protein = elute faster - gel filtration
A
You know which fractions (elution volume) correspond to a specific mass
Can load sample in high salt buffer - therefore can perform straight after protein precipitation
16
Q
Protein of interest
A
After GFC, take a sample from each fraction and perform a Western blot for your proteins of interest
GFC resins - designed to have pores that allow separation of proteins within a particular mass range
Can check mass range of sample using SDS-PAGE, then choose resin
17
Q
Column calibration
A
A range of protein standards are used to calibrate a column
Protein elution monitored by UV Abs and elution vol matched to mass
Each lane corresponds to a fraction collected during gel filtration
18
Q
Protein complexes -
Factors Affecting Separation in GFC
A
Protein complexes - Complexes generally intact when proteins in native state
Factors Affecting Separation in GFC
Size/mass of protein - molecular radius, proportional to mass
Shape of protein (e.g. globular vs fibrous)
Length of column – longer columns give better separation
Amount of protein – too much protein can cause broad elution peaks
Resin material (e.g. pore size)
19
Q
separates proteins based on charge
A
charge comes from ionisation of amino acid side chains
At physiological pH, Glu and Asp lose H+ (acidic side chains)
At physiological pH, Lys and Arg gain H+ (basic side chains)
20
Q
7 amino acids have side chains that can be ionised - ion exchange Chrom
A
Aspartic acid (pKa 3.9)
Glutamic acid (pKa 4.3)
Tyrosine (pKa 10.1)
Cysteine (pKa 8.3)
Arginine (pKa 12.5)
Lysine (pKa 10.8)
Histidine (pKa 6)
pKa value = acid dissociation constant (pH at which 50% ionisation occurs)
At pH below pKa - side chain accepts H+ (protonated)
At pH above pKa - side chain loses H+ (deprotonated)
21
Q
Isolectric point
A
Overall protein charge - determined by the proportion of acidic and basic amino acids
Isoelectric point (pl) - pH at which protein has no net charge
pH below isoelectric point = net positive – decreasing pH (protonation)
pH above isoelectric point = net negative – increasing pH (deprotonation)
If you know pI of protein, then you can adjust the pH to alter net protein charge
22
Q
Exchange resin - IEC
A
Cation exchange resin - Binds to positively charged proteins (‘cations’)
Resin has a –’ve charge (e.g. CM-cellulose, S-Sepharose)
Anion exchange resin - Binds to negatively charged proteins (anions)
Resin has a +’ve charge (e.g. DEAE-Sepharose, Q-Sepharose)
To purify target protein - need to use appropriate buffer pH and the correct resin
Want target to bind to resin
Bound proteins are then eluted with buffer containing increasing salt concentration
23
Q
Hydrophobic interaction chromatography
A
interaction between hydrophobic patches on protein and resin coated with hydrophobic material
24
Q
In aqueous solution - HIC
A
In aqueous solution - proteins have hydrophilic surface with hydrophobic patches
water forms a ‘shield’ around the protein surface – hinders hydrophobic interactions
HIC - sample is prepared and loaded onto column in high salt buffer
Salt - displaces water and exposes hydrophobic patches
For protein binding to the resin, salt concentration is inversely proportional to protein
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Hydrophobicity - HIC
For protein elution, a decreasing salt gradient is used.
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Factors that may impact elution
Choice of salt in buffer (Hofmeister series)
Include non-ionic detergents (reduce hydrophobic interactions)
Reduce temperature
Change pH – proteins are least soluble (most hydrophobic) at their isoelectric point
27
Isolectric focusing
At a specific pH proteins have an overall neutral charge
Isoelectric focusing
Protein is loaded onto a gel with stable pH gradient
An electric field is then applied – proteins migrate based on their charge
Proteins will migrate along the pH gradient until they reach their isoelectric point
At high pH - protein is -’vely charged
At low pH - protein is +’vely charged
phosphorylation - adds a negative charge to proteins and alters migration in IEF
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2-Dimensional Electrophoresis- protein analysis
First separation - based on charge (IEF) -follow with SDS-PAGE
Second separation - based on molecular weight (smallest move fastest)
Can perform Western blotting after 2D electrophoresis to detect protein of interest
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SDS-PAGE protein analysis
Separates proteins based on their size
Need to unfold proteins (denature)
Use sodium dodecyl sulphate (-’ve charge) and DTT (reduces disulphide bonds) and heat to 95C 5min
Protein samples are loaded into wells of gel and electric current is applied
-’vely charged proteins migrate towards positive electrode
After electrophoresis, proteins can be visualised using Coomassie Blue
Large proteins remain near top, smaller proteins migrate to bottom - Need to include molecular weight marker on gel
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Checking Expression and Purity of your Protein
Protein assay to measure protein concentration - then analyse purity using SDS-PAGE
Lysis buffer and wash steps can be modified to improve yield and purity of your target protein
Ireduce non-specific binding to your affinity resin
For protein binding - salt concentration is inversely proportional to protein hydrophobicity
For protein elution - decreasing salt gradient is used.
31
Factors that may cause low proteins concentration
Poor protein expression in bacteria – optimise growth/IPTG
Inefficient lysis – try other methods/combinations
Inefficient purification – reduce detergent/salt
Inefficient elution – optimise
Protein is insoluble – optimise expression conditions/use mammalian host
Protein degradation - proteins are prone to degradation throughout the process
Minimising Proteolysis - Major cause of protein degradation are protease enzymes released during cell lysis
Low temperature, Work quickly, protease inhibitors, chelators, SDS-PAGE
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Factors that affect protein migration - purity factors
Proteins generally migrate based on mass - but can migrate based on size
Large post translational modification (e.g. ubiquitylation and glycosylation) cause proteins to migrate at higher mass
Small PTMs (e.g. phosphorylation) generally don’t affect protein migration
If proteins are not fully denatured, they might migrate as complexes (e.g. dimer)
Inefficient reduction of disulphide bonds
High content of basic amino acids can affect migration
33
Immunoprecipitation
precipitating a protein out of solution using a specific antibody to protein of interest
agarose beads coated with protein A/G which bind to antibodies with protein of interest
Beads insoluble and heavy - precipitate using centrifugation or magnetism
isotype control antibody (gold standard) - primary antibodies that lack specificity to the target -help differentiate non-specific background signal from specific antibody signal.
% input method = sample / input
Fold enrichment = sample / noise (control)
34
Co-immunoprecipitation (Co-IP) & RNA-immunoprecipitation (RIP
Use - Analyse protein–protein interactions
Sample preparation (non-ionic detergents - e.g., NP-40, Triton X-100)
Pre-clearing (just beads)
Antibody incubation (target antibody or isotype control antibody)
Precipitation of protein/protein complexes
Washing
Elution and analysis of precipitate (low pH or high salt solution)
Analysis - SDS-PAGE, Western blotting, Mass Spectrometry
35
RIP
Proteins bound to RNA
Use - Study the physical association between individual proteins and RNA molecules in vivo.
Classes
Native – used to identify RNAs directly bound by the protein and their abundance in the sample.
Cross-linked – used to precisely map the direct and indirect binding site of the RBP of interest to the RNA molecule.
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Proteins bound to DNA-Chromatin-immunoprecipitation (ChIP)
Proteins bound to DNA
Use - Investigate regions of genome associated with a specific protein
Steps
Cross-link and harvest cells (Cross link DNA & protein) - Cross linking agent = formaldehyde
Cell lysis & chromatin fragmentation
Immunoprecipitation
Wash, elution and cross-link reversal (Remove DNA from protein)
DNA cleanup and analysis of DNA - PCR, qPCR, microarray, sequencing
37
Controls
Input DNA - A chromatin sample processed parallel to the other samples but lacks the IP step.
No Ab control - A chromatin sample processed parallel to the other samples but immunoprecipitated without specific antibody
Isotype Ab control - A chromatin sample processed parallel to the other samples and immunoprecipitated with an isotype Ab control (IgG or IgM)
Histone H3 antibody - A chromatin sample processed parallel to the other samples and immunoprecipitated with anti-H3 ab
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Antibodies
must be highly epitope-specific to protein of interest in their native chromatin states or possible cross-linked formation.
anything associated with chromatin can be ChIPed, if an antibody can be raised.- commercial kits
protein of interest is immunoprecipitated together with the crosslinked DNA - Decrosslinking & Proteinase K digestion then DNA purification
ChIP-PET (paired-end tag sequencing to determine long-range interactions)
ChIP-DSL (DNA selection and ligation strategy using specific oligos)
ChIP-BA: combining ChIP with bisulfite genomic sequencing for analysis of DNA methylation
Methyl-DNA immunoprecipitation (MeDIP)
PCR and qPCR - Identify DNA regions associated with the protein of interest - however primers bias toward sequences of interest.
ChIP-ChIP = DNA microarray (genome mapping)
Protein of interest is selectively ChIPed.
ChIP-enriched DNA amplified by PCR & fluorescently labelled.
An aliquot of purified input DNA is labelled with another fluorophore.
2 samples are mixed & hybridized onto a microarray.
Binding of the precipitated protein to a target site is inferred when intensity of the ChIP DNA (cy5 label) significantly exceeds that of the input DNA (cy3 label) on the array.
39
Applications
DNA sequences occupied by specific protein targets
The binding sites and distribution of a particular protein, such as transcription factor, throughout the entire genome, under specified cellular conditions
Gene transcription and RNA polymerase activity
Complex DNA/protein interactions underlying disease phenotypes
Modification to protein, such as histones, that many influence chromatin structure and gene expression
Nucleosome architecture and regulation of chromosomal maintenance
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Types of Data
qualitative – nominal scale (categories - can be converted into frequencies)
ranked – ordinal scale (sequence order)
quantitative – various scales (‘real’ numbers - nonparametric)
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Quantitative data
Discontinuous (discrete scale) - Obtained by counting (integers)
Continuous (interval scale) - Obtained by measurement (Sf or Dp)
Derived data – calculated from direct measurements (e.g. ratios, percentages, rates, etc
42
Stats
Accuracy - Closeness of measurements to true value (mean)
Bias - systematic or consistent deviation of measured values from the true value
Precision - Closeness of repeated measurements to each other (variability)
Graphs
X-axis = Independent variable (changes in experiment)
Y-axis = dependent variable (measured)
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Mean, SD , CoV
Median - Midpoint of a data set, ranked from low to high (tendency)
less sensitive to outliers - central point of data (unaffected by variation/ skewed date)
keeps data at normal distribution
Mean - average value of the data
average deviation from the mean - Would use all data values - but would be 0 as deviation below mean would cancel out that above the mean
95% of population lies within 2 SD of the mean
true negative population = mean + 2SD = 97.5% - assuming normal distribution
negative population in first log decade (10-1)
eliminates true negative but detects weak positives
Standard deviation - Standardised measure of spread in a data set (The positive square root of the sample variance = units same as mean)
- lower SD = more precise
Coefficient of variance (CV or CoV) - sample standard deviation in proportion to the sample mean
CoV = (SD ÷ mean) x 100
Low CoV = higher precision
Normal distribution - Mean, median, mode are all equal, bell shaped curve & 95% of population lies within 2 SD of the mean
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Hypothesis
Alternative (H1) - There is a difference or relationship or association
2-tailed = difference could be in either direction (Gold Standard - testing for a difference vs. no difference)
2-tailed ‘working hypothesis’ - “There is A difference between the test results using method A and method B”.
2-tailed null hypothesis - “There is no difference between the test results using method A and method B”.
1 sided testing (1 tailed) - 2 possible one-sided (one-tailed) working hypotheses:(higher or lower)
P value - the probability that the null hypothesis is true.
If P is less than 0.05 - reject H0 and accept H1
If P is 0.05 or greater - accept H0 ( random chance)
45
T Test
Comparing 2 groups of data
Based on Probability - accept or reject the null hypothesis →Reject null hypothesis if P <0.05 (<5%)
continuous quantitative data
T statistic - higher value = more likely the two sample means are to be different
Paired t test - compares means from the same sample group before and after a specific intervention, or period of time.
Unpaired t-test - 2 completely separate/ unrelated groups
46
ANOVA
ANalysis Of Variance
Comparing more than 2 group
Key advantage - only one test, rather than multiple tests → Reduce number of tests = remove number of false positives (type I errors)
Box and whisker diagrams - 2 or more groups, single measurement
Results - quote F statistic and the p value
Continuous quantitative data
Data must be normally distributed & Variances must be homogeneous (equal level of variability in each group)
Samples must be independent of each other (not paired)
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Variances
Why do we use unequal variances?
To reduce number of stats tests as much as possible - better to assume unequal variance rather than do yet another test for equality of variance
Interpreting stata results
State test used - stat test performed to investigate…..
state output values (mainly P value)
state if we accept H1 or H0
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Experimental design
Why we use Experimental design - investigate. Hypothesis,m plan, power, data, result
Experiments - controls, variables, replicates
Type of data to collect - quantitative, Ordinal scale, Qualitative (nominal) variables, stats, graphs
Factorial design – alternative to changing one factor at a time.
Less factors = less repeats
full factorial method - in which all permutations are carried out
Fractional factorial methods
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Experimental d - manipulation
Manipulative = one or more factors are deliberately altered
Purpose - explore cause and effect
treatments = sets of conditions
blocks = groups of replicates subjected to the same treatment
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Experimental d - observations
Observational = Investigate links between variables of interest occurring in natural conditions
Purpose - comparisons between natural situations
treatments = sets of natural measurements
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Type I vs Type II errors
Standard curve
Left 0.05 = type II error
Right 0.05 = type I error
Rest = 95% (power, 1-β)
the lower the value of P - the more confidence we can have in our decision to reject the null hypothesis and accept the alternative hypothesis.
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Type 1 error
Type I error rate (α) - statistical significance level (0.05) (false positive)
P value must be lower that 0.05
Reduce Type I error probability- set a lower significance level (but increases type II risk)
Worse to make
Accept null when should reject
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Type 2 error
Type II error rate (β) - probability that a false H0 is retained (false negative)
failed to find adequate evidence for a difference
Higher power = lower type II error rate (increases type I risk)
Reject null when should accept
54
Power
Power = the probability of correctly rejecting a false null hypothesis / of finding a significant difference
Avoid type I or type II errors
Reduce unnecessary risks
As standard deviation increase, power decreases
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Effect size
Effect size (ES) - measures the strength of the result (between 2 variables)
solely magnitude-based - does not depend on sample size.(but needed to determine N)
We need to know if ES found is applicable for population (relates to p value)
derived from - Pilot study results, Published findings from similar studies, guesswork
Stakeholders
Cohen’s d - standardised mean ES used when comparing 2 means in a standard deviation unit (comparative)
bigger effect = less samples
d = (mean2 - mean1) / st.dev pooled
56
Sample size
Sample size (N)
Increasing the sample size increases power - not a linear relationship
often what is required to calculate
might be restrained or fixed due to subject availability or resource limitation
Do not waste resources or time
Too many samples is better than too few
Sample too small - unable to reject null hypothesis
Sample too large - some subjects have been unnecessarily exposed to risk of harm
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Probability
Probability (ɑ, P)
P‐value - relates the likelihood that what you found is not due to chance.
dependent on sample size
Best practice - report effect sizes, p-values and conduct a power analysis
Power calculation - knowing any 3 these values allows calculation of the other
α and power are usually set
58
Power analysis
Check slide 18
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Enzyme assays 1
Calorimetry - measuring the amount of heat released or absorbed during a chemical reaction. exothermic (releases heat) vs endothermic (absorbs heat)
Spectrophotometry - measure light absorption or the amount of chemicals in a solution
Dry phase/ solid phase - portable, easy to use and point of care enzymatic assay kits (Fully quantitative or semiquantitative allows estimation of the content in a sample) → chemical (adsorption) bonds or covalent bonds
Manometry - measure enzyme activity if one of the components is in gaseous form
samples and reagents are placed in separate compartments and mixed at defined time period and the reaction can be followed as the reaction proceeds.
end point and kinetic assays can be performed.
E.g Oxygen consumption is measured in glucose oxidase
Radiochemical methods
Radioactively labelled substrate - used to follow the enzymatic reaction
Highly sensitive where picomolar concentration of reactants and products can be measured
Common radioisotopes - 3H (tritium), 32P (Phosphorus), 35S (Sulphur) ,131I (Iodine).
enzymatic reaction is performed for a defined period and quenched using a reagent
substrate is then separated from the product using electrophoresis or chromatography
radioactive fraction of the product or the substrate is used to estimate activity of the enzyme
60
Enzyme assays 2
Electrochemical methods
Potentiometric Techniques – Electrical potential generated is dependent on the concentration of the substance in solution
Polarography/Voltammetry- Increased Voltage is applied between 2 electrodes immersed in a test solution and the change in potential is measured.
composition of test solution determines current which flows at each instance
Enthaplimetry - measures the enthalpy change during the course of a reaction
Advantages - Sensitive, Easily adapted for various applications - don't need to know substrate or product
Disadvantage - interference - e/.g tags, fluorochromes
Requirements - Extremely accurate thermostatting or excellent insulation
Spectrofluorimetry
Formation of product or reduction of reactant concentration - measured by attaching a moiety that fluoresce at defined wave lengths
At low concentrations - fluorescent intensity is related to the intensity of light (fo) of appropriate wavelength by the relationship
If- Io x 2.3Eclq
E is the molar absorption coefficient
c the molar concentration
l –length of the light path
q= quantum efficiency (number of quanta fluoresced / number of quanta absorbed)
Example - Dibutyryl Fluorescein (non-fluorescent) + Lipase activity → Fluorescein (fluorescent)
not bulky
chromatography column to separate and purify
Does not interfere with reaction or product
natural well established small fluorophores
61
Animal sources- therapeutic enzymes
Lipase (lipid breakdown) - Animal Pancreas
Trypsin (protein breakdown) - Ox Bile
Urokinase (inactive plasminogen into active plasmin) - Human Plasma / Cow Urine
Lysozyme (cleaves peptidoglycan in bacterial cell walls)- Egg
Adenosine Deaminase (deaminates deoxyadenosine) - Bovine intestine
Pepsin (protein breakdown) - Hog Pancreas
Dornase α (recombinant human cells) - mucus breakdown
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Plant sources - therapeutic enzymes
Plant sources
Papain (Carica Papaya) - protein breakdown
Nattokinase (Natto) - dissolves blood clots
Amylase (Malted barley-Hordeum vulgare) - starch, carbohydrate breakdown
Bromelain (Ananas Comosus) - reduces swelling
63
Bacterial- therapeutic sources
Beta lactamase (Staphylococcus sp.) - antibiotic resistance
Staphylokinase (staphylococcus sp.) - dissolves clots (plasminogen to plasmin)
Rhodanese (Sulfobacillus sibiricus) - detoxify cyanide
Streptokinase (hemolytic streptococci) - dissolve clots
L-asparaginase (E.coli) - L-asparagine → L-aspartate + ammonia
Collagenase (Clostridium histolyticum) - collagen degradation
Amylase (Bacillus sp.) - starch, carbohydrate breakdown
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Microbial sources (prefered workhorse)
Microbial sources (prefered workhorse) - bacterial (and fungal)
Cheaper to Produce
Content of enzyme estimated & controlled
Reliable supply for Raw material of constant composition
Other sources contain more harmful phenolic compounds,
endogenous Inhibitors and proteases
65
Disease enzymology
Enzymes in plasma are measured
Plasma specific enzymes - e.g fibrinolytic enzymes
Secreted enzymes - secreted outside of cell (e.g pancreatic enzymes)
Cellular enzymes - normally should be within the cell
half-life dependent on inactivation or removal :
AST = 17 hours
CPK = 15 mins
Factors that increase Plasma enzymes - cell damage, increased proliferation, increased enzyme synthesis, decreased clearance
Level of increase of plasma enzyme based on - degree of damage, original intracellular levels & amount of tissue affected
66
Enzyme profiles to diagnose diseases & assess organ function
Enzyme profiles to diagnose diseases & assess organ function
Identify where the enzyme came from - localise site of damage/ source of enzyme
Measure organ specific enzyme
Measure isoenzyme - catalyse the same reaction but have different primary structure, physical & chemical properties
Isoenzymes
Measurement - electrophoresis, immunoassay. Thermostability, Inhibitor sensitivities, Modified substrates, coenzyme analogues
LDH isoenzyme detection = electrophoresis
LDH1 &2 in heart muscle & RBC, LDH5 in liver and smooth muscle cells.
Creatine Kinase (CK) or Creatine phosphokinase (CPK) -Electrophoresis & Column Chromatography:
CK1 (BB), CK2 (MB),CK3 (MM - 100% of serum CK)
67
Automation
Automated enzyme analysis interference - Haemolysis, turbidity and lipaemia
Inserted lag phase & automatic blanking
Automated analyser - Ideal for kinetic studies but must be computer controlled → disposable, fast, discrete analysis
Continuous flow = measures start point and predetermined time of end point, then plots curve
Automation advantages - Improved efficiency, fast, precise, smaller samples, less errors, continuous, less manual labour
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Flow cytometry
Flow cytometer - an instrument capable of simultaneous measurements of size/ granularity /fluorescence of a single cell.
measurements on a per cell basis at rates typically in the order of 500 to 4000 cells per second in a moving fluid stream
Flow cytometer vs cell sorter - Flow cytometer can only analyse cells, cell sorter can sort AND analyse cells
Requirements
Fluidics - The fluidic system is used to deliver the particles individually to a specific point imtersetcd by a laser
Optics - Consists of an excitation source and data collection optics
Electronics - conversion of optical signals into electronic signals for data analysis
Analysis and Display
Gating - the ability to select a population for analysis
Intrinsic - No reagents or probes required (Structural)
Cell size (Forward Light Scatter)
Cytoplasmic granularity (90 degree Light Scatter)
Pigment content e.g. Haemoglobin
Extrinsic - reagents required (fluorochrome)
Structural - DNA content, DNA base ratios, RNA content
Functional - Surface and intracellular receptors, DNA synthesis, DNA degradation (apoptosis), Cytoplasmic Calcium, Gene expression
Forward scatter - size (bigger the cell the larger the forward Scatter)
Side scatter - granularity (internal structures & cell surface characteristics)
frequency histogram - direct graphical representation of the number of events for each parameter analysed (1 dot = 1 cell)
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Fluidics & electronics
Fluidics
Sheath fluid – filtered isotonic saline (always flowing)
Cells line up in centre of the tube due to low pressure (pressurised air forces liquid out)
Troubleshooting - Cracked sample tube - air can’t force out liquid, so use new tube
A stream velocity of 10m/s is required →10 micron particles will then traverse their own diameter in 1 microsecond.
Electronics
preamplifier - smoothen pulse to make louder
logarithmic or linear scale
Analog to digital conversion - converts voltage to binary for analysis on computer
Flow cell - quartz glass - transparent to all light wavelengths (& cleanable)
Spectral overlap
PMT tuned to detect light in certain wavelength →FITC emits multiple wavelengths
compensation
first log decade is where negative signals seen
auto compensation software - input colours wanted and works out values
Beads should not go above unlabelled beads
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Excitation source
Flow cytometry optics
Excitation source
Arc lamp - glass envelope containing a gas or vapour at high pressure
initial high voltage spark between 2 electrodes creates a plasma arc → maintained by the application of high current at a low voltage
Prone to flicker and average life of arc lamps is short
Laser - coherent, monochromatic, plane polarised, intense beam of narrow light
Reacts with fluorochrome - The light emitted is reflected along the tube
Brewster window - small quantity of polarised light passes through
Plasma tube - contains gas under pressure which fluoresces under the application of current
When these photons strike an atom in an excited state they release another photon of the same wavelength
Common lasers - diode 636nm & argon ion
Light released is larger wavelength than input
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Flow optics
Fluorescence - Occurs when a molecule is excited by light of one wavelength returns to the ground state by emitting light of a longer wavelength
Application of fluorochromes
cells can be stained (the cell will bind a Fluorescent Dye)
And/or a fluorochrome conjugated with an antibody in an amount proportional to the quantity of the Binding Constituent (eg, DNA, RNA, Surface antigen). The cell’s emitted fluorescence intensity will then be proportional to the fluorescing cellular constituent
Common fluorochromes
FITC - Bright, Absorption maximum close to emission lines from both the argon laser and a mercury arc lamp (rare components are brighter)
R-phycoerythrin (PE) - Can be excited at 488nm so only one laser required
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Flow cyt mechanism
Filters and mirrors
Dichroic mirrors (beam splitters) - Allow light of a certain wavelength to be reflected while the remaining wavelengths can pass through
Shortpass filters - Allow light below a specified wavelength through
Bandpass filters - Only allows a specified range of light wavelengths through
Longpass filters - Allow light above a specified wavelength through
Detector types - data collection
Photodiodes - Newer technology, High efficiency for visible spectrum, No adjustable gaim, Requires cooling
Photomultiplier Tubes (PMT) - Detect Light/ fluorescence & are Most common detector in flow cytometry → Old well characterised technology & Cheap
Mechanism - photon exchanged for 1 electron, then electron number doubles at dynodes until reaching anode (often 8 fold - 1 electrons, 2, 4, 8 etc)
Amplify signal so good for the detection of weak fluorescence
Adjustable sensitivity but poor efficiency in red (>650nm)
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3 colour lymphocyte immunofluorescence (LIFT)
Crossmatch - mix typed lymphocytes with patient serum
combines tests - HLA I, HLA II, antibody (IgM, IgG) for less time
Anti CD3/PE - only binds to T-Cells
Anti CD19/PerCP - only binds to B-Cells
Anti IgG FITC (or IgM / combo of both)
T & B cells - same side and forward scatter - can be gated
analyse results based on PE, PerCP or FITC
PE = T cell
FITC = antibody detects both T & B cells
PerCP = B cell
can detect hyperacute rejection
Multiple individual typed cells required so slow
can count 10,000 cells - sensitive & repeatable
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Multi-Colour Flow Cytometry (MCFC)
More Detectors
More Lasers - intersect at different points at different times or filtered through a single point at the same time
Increasing the number of lasers = increases the number of fluorochromes= increases the number of detectors
Example MCFC - dako cyan
Disadvantages - More cost & more complications
Advantages
More accurate population identification - e.g CD markers
Use smaller specimens as more parameters are available to test in one tube
Save time and reagents as fewer tubes are required to be tested
Capable of collecting large number of events more efficiently
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Luminex Technology - multiplex
Luminex Technology - multiplex
Beads are incubated with sample then washed before addition of PE reporter
96 well plate capability so high throughput is possible
Allows multiple analyses in one tube (Maximum 100) - no cells just beads
microspheres (beads) to which reagents can be bound to
5.6 μm polystyrene microspheres
Each microsphere is dyed with a combination of red and infra-red fluorochromes - allows the definition of 100 different beads
2 lasers used - precision fluidics aligns microspheres in a single file, then passes them through the lasers one at a time
First laser excites molecular tags - reaction measured by fluorescent intensity in real time (green)
Second laser excites microsphere - fluorescent intensity of microsphere identifies the reaction (red)
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Cell sorter
Cell sorter - a flow cytometer with the added ability to physically separate out a population described by a gate
Dako mo flo - Analyzes and sorts cells at 70,000 cells per second (expensive)
Coincidence - At high sample event rates the possibility exists that cells not fulfilling the criteria may be sorted
occurs if 2 or more cells are detected in the time frame of droplet formation
Anti-Coincidence gating can be used to prevent this
It works by creating a time window around the particle of interest relating to droplet formation
If any other partial is detected in this window then the stream is directed to waste
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Cell sorter
Accuracy
Accuracy of droplet charging
droplet formation is a stable process - however can be affected by sheath temperature or sheath pressure (form faster of slower)
This may lead to the charging pulse not being delivered to the correct droplet
To overcome this it is common to charge more than one (2 or 3) droplet
This can decrease purity without anti-coincidence gating (enrich mode), or decrease yield with anti-coincidence gating on
Phase gating - Determines if cell is in the centre or outside quarters of the droplet window
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Flow cytometry evolution history
1934 – Moldovan described a method for counting cells automatically on a microscope
1949 – Coulter described the 1st automated blood counter based on conductivity
1953 – Crosland-Taylor utilise laminar flow and hydrodynamic focusing for particle alignment
1965 – Kamensky developed the use of spectrophotometry to quantitate cellular constituents, introducing multiple cellular measurements
1965 – Fulwyler developed the first cell sorter, allowing the physical separation of cells based on multiple parameters
1980 – Development methods and fluorescent dyes
1990 – Benchtop flow cytometers capable of 5 parameters at 10,000 cells/sec introduced size, cell content + 3 fluorochromes)
2001 – Flow cytometers capable of 11 parameters using 3 lasers at 25,000 cells/sec introduced (size, cell content + 9 fluorochromes)
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HLA antibody detection
Flow methods use beads coated in HLA or HLA typed cells
Flow Cytometer (beads & cells) - slower, 8 beads
Luminex (beads) - faster, up to 100 beads
IgG - Complement fixing antibody → hyperacute rejection (destroy tissue)
IgG against HLA I & HLA II show poor prognosis
IgM - Non-complement fixing
IgM associated with naïve CTL (CyA sensitive)
Patients with IgM HLA antibodies - may also have IgG antibodies with the same specificity
Screening bead - mixture of most common HLA antibodies (general)
ID test - 1 HLA antibody per bead (specific)
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HLA antibody detection - luminex
Principle of luminex - only beads, faster
HLA antigens bound to multiple microspheres - Up to 100 beads can be used.(all HLA in 1 tube)
Dark - so UV doesn't bleach dyes from beads
Temperature - reaction occurs at appropriate speed
Agitation - beads will clump together if settled, so antibodies can’t enter
Opsonize - if Patient serum contains complementary antibody -
Wash - remove unbound antibodies
gates - set to detect beads based on fluorescence
100ul anti human IgG-PE - added to mixture so bind if antibodies present on beads
Reporting laser - any PE bound (i.e bead colour, red vs infrared)
Beads are negative if no PE fluorescence
Microsphere ID Laser - which bead is it (fluorescence as antibody present - i.e HLA A3 is positive as anti-HLA A3 present
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HLA typing
CD34 - Immature cell line marker for HSC
Present on 2-4% of all normal marrow mononuclear cells
Flow methods - use a mix of anti-CD34, anti-CD45 & fluorosphere to generate an absolute count
CD45 - All WBCs
CD34 - Immature cell line marker for HSC →Gate to detect CD34
Flow cytometers can’t work out absolute counts - volume used unknown
Fluorospheres (158 per microlitre) - added to obtain absolute count
Making flow cytometer quantitative - less problems and increases accuracy
CD34 enumeration
Absolute Count (cells/μl) = (No. CD34 cells + Fluorospheres Conc) / Number of Fluorospheres counted
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HLA typing
By luminex
HLA Typing by Luminex
PCR-SSO - sequence specific oligonucleotides
Amplification of HLA gene locus of interest with biotinylated primers
oligonucleotide placed onto bead (24-40bp) - specific to one HLA gene (if it binds gene is present)
Denaturation of amplified product
Hybridisation of amplified product with Oligonucleotide probes bound to beads
Addition of Streptavidin/PE reporter - 4 streptavidin bind to 1 biotin laced DNA
Computer analysis of positive results leads to assignments
2 signals - PE for gene presence, bead1 with HLA I
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Complement dependent cytotoxic test (CDC)
low resolution to serologically identify Digits 1 & 2 of HLA nomenclature.
It uses HLA-specific antibodies, from characterised allospecfifc antisera or monoclonal antibodies that bind to the HLA molecules expressed on the surface of patient or donor cells.
Peripheral blood lymphocytes are separated into T & B cells, allowing detection of Class I or Class II antibodies
HLA class I expressed on the surface of all nucleated cells
HLA class II expressed only on B cells, APCs & activated T cells.
mineral oil - prevent liquid evaporation (very small volumes used)
Antibodies- either a known (typing) or unknown (screening) specificity
Donor = crossmatch
Known HLA = screening
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Complement dependent cytotoxic test (CDC)
Fluorescence
Lymphocytes stained with a fluorescent dye (e.g ethidium bromide) which is able to distinguish live cells from dead cells are then added and the reaction occurs for 30 minutes at 22oC, after which opsonization occurs if antibodies and lymphocytes bind and fluoresce.
After these 30 minutes rabbit complement and PI (Pyridinium iodide) are added and the reaction resumes for an additional 60 minutes at the same temperature.
Complement initiates the classical complement cascade and MAC formation if the antibody recognizes the antigen on the cell surface
Bind to fc region of antibodies
Allows PI into the cell and red fluorescence - green fluorescence means the Pt is not inside the cell,I.e alive
Ink and EDTA are added
EDTA stops the complement reaction
ink provides a dark background for fluorescence
The percentage of cell death in each reaction is noted and each well given a score, with multiple reactions analysed to determine a patient or donor’s phenotype.
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Platelet antigens - ABO, HLA I, HPA
Platelet specific - human platelet antigens (HPA)
Bi-allelic co dominant
A is most common variant, B is rarest variant
Single point mutation - Difference between A & B
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NAITP - Neonatal alloimmune thrombocytopenia
NAITP - Neonatal alloimmune thrombocytopenia
Reduced platelet count at birth
Symptoms - Petechiae rash, bruising, intracranial haemorrhage, low platelet count (thrombocytopenia)
Relatively common - 1/1000 pregnancies
Can occur in first pregnancy
85% caused by mothers anti HPA-1a - antibody against most common variant
HLA DRB3*0101 association - 1:3 chance of forming antibody
Unusually unexpected - no antenatal screening programs
Not associated with anti-HLA
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Platelet immunofluorescence test (PIFT)
Use fathers platelets & mothers serum
father is source of platelet antigen being reacted against
Gate on platelets based on low forward scatter (platelets are small) & low side scatter (limited granularity)
Analyse gated region for FITC fluorescence
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Dna analysis
DNA analysis
One of the first application of flow cytometry
Malignant cells are often aneuploid - abnormal number of chromosomes
More chromosomes = worse prognosis, resist treatment and increased malignancy of cancer
DNA content of a tumour may be expressed as the DNA index
DNA index - ratio between the DNA content of a tumour cell and that of a normal diploid cell
Diploid = 2n (2 x no. chromosomes)
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Dna analysis method
PI binds stoichiometrically - number of molecules of probe bound = number of molecules of DNA
Can’t enter cell through cell membrane
Detergent (triton-X) - disturbs membrane - allows PI inside (fluorescence)
Measure cell counts against PI fluorescence
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Cell cycle analysis
Application of flow cytometry - quantitation of cellular DNA
Still method of choice - fast, accurate determination of cell cycle distributions
4N = duplication, return to 2N following mitosis (M)

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Immunophenotyping
Immunophenotyping
Help diagnose leukaemia - presence or absence of cell surface markers
Identify expansion of type of cells - tumour identification
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Immunology - Chronic granulomatous disease
Phagocytes can’t form oxidative burst
Phagocyte NADPH oxidase - responsible for generation of oxidative burst
Inactivated by genetic mutation
Phagocytes can’t form oxidative burst
Symptoms/ susceptibilities
Pneumonia, abscesses, suppurative arthritis, osteomyelitis, bacteremia, superficial skin infections - e.g cellulitis, impetigo
Flow diagnosis
Incubate whole blood with PMA & DHR-123
PMA stimulates neutrophils to undergo oxidative burst
DHR-123 - oxygen sensitive dye that fluoresces at 535nm → colourless (unless oxygenated via oxidative burst)
Gate on FS/SS for Neutrophil - high side scatter
Peak = positive for oxidative burst
Analyse Neutrophils for FL1 fluorescence
first peak - cells below 101 - no oxidative burst
second peak - positive burst over 102
red area - unclear data, potentially due to mis-compensation, laser going off, blockage in system
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Transfusion related lung injury (TRALI)
Transfusion related lung injury (TRALI)
Severe type of non-hemolytic transfusion reaction
Donor antibodies react with patient
Plasma rich = higher chance of donor antibodies (foreign)
Acute respiratory distress - will pass with ventilation as donor plasma is removed from system
Cause - unclear, associated with antibodies to WBCs
HLA I & HLA II antibodies
Anti Granulocyte antibodies (HNA) - donor antibodies against neutrophils
Principle - modification of LIFT
Gate on lymphocytes & granulocytes - can test patient against donor plasma
Incubate with anti-human IgG / FITC
Spike for FITC = antibody can recognise neutrophils (TRALI occurs(
Analyse lymphocytes & granulocyte populations for FITC fluorescence
