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Clinical Chemistry > Week 11 > Flashcards

Week 11 Flashcards

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1
Q

What is electrophoresis

A

-separation based on charge
-proteins will have a charge because of ionization of functional groups
-NH2, COOH, R group
-current is applied
-charged particles migrate toward opposite of charged molecule
-mobility of particles is determined by the environment
-proteins have different velocities

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2
Q

What are the components of PE

A

-power supply and chamber with 2 platinum electrodes
-basic buffer at pH 8.6, uses wicks to maintain contact with hydrated support medium agarose gel or cellulose acetate
-reference sample with normal and abnormal
-pt sample
-pe chamber
-detection and quantitation - fixing, drying and staining
-chamber lid prevents solvent evaporation due to heat
-Anions go to positively charged anode and cation go to negatively charged cathode

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3
Q

What is iontophoresis

A

migration of small ions

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4
Q

what is zone electrophoresis

A

-charged macromolecules migrate in a porous support medium like cellulose acetate, agarose gel film and polyacrylamide gel
-macromolecules can be proteins in serum, CSF and urine

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5
Q

What is an electrophoretogram

A

-generated by zone electrophoresis
-creates zones or fractions of macromolecules
-physically separated from each other
-can be homogeneous (no separation step) or heterogeneous (needs a separation step)

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6
Q

What is an ampholyte

A

-has acidic and basic group
-used to establish stable pH gradient as they can act as an acid or base depending on the pH

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7
Q

What are the factors that affect mobility

A

-Net charge on the protein
-Size & shape of the molecule
-Ohm’s Law & power supply
-Heat production (heat produced when current flows through a medium that has resistance)
-Support media
-Time
-Ionic strength of buffer
-Electroendosmosis

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8
Q

how does the “net charge on a protein “ affect mobility

A

-pI = isoelectric point (IEP)
-if pH < IEP the protein accepts H and has a overal positive charge because cations migrate to cathode (-)

pH = IEP ; net zero charge and no migration

pH> IEP ; proteins will donate H, have an overall negative charge because anions migrate to the anode (+)

the pH of the buffer around the proteins controls/alters protein charge and influences its motility

The greater the net charge of a protein, the more it is attracted to the oppositely charged electrode and the faster it moves

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9
Q

how does the “Size and Shape of Molecule “ affect mobility

A

-protein motility is counteracted by frictional resistance
- based on the ionic radius of the particle
-smallest molecules move the farthest

Albumin -smallest and fastest
Globulins- large asymmetric globular and moves slower

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10
Q

how does the “Ohm’s Law & power supply “ affect mobility

A

Electrophoresis is governed by Ohm’s Law: V = I R
V= electromotive force (in volts)
I = current (in amperes)
R = resistance (in Ohms)

Gel is the resistor
power supply determines current (I) and voltage (V)

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11
Q

how does the “Power Supply” affect mobility

A

-keep rate of migration constant
-distance of migration (mobility) is directly related to the voltage and time (length of run)

low voltage = low mobility and vice versa
-a high voltage reduces the time to produce zone separation
-if current is constant, voltage will increase as resistance goes up
-high voltage leads to heat production because the current is moving through resistance

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12
Q

how does the “Heat Production” affect mobility

A

-high voltage results in increase of thermal agitation of the dissolved solute (proteins) causing a decrease in resistance to migration and increase in current
-increase of evaporation of water from buffer which increases ion concentration of the buffer . This increases migration rate and may cause solutes to distort because of diffusion of seperated zones

-constant current keeps heat and solute diffusion low, as electrophoresis progresses a decrease in resistance also decreases the voltage

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13
Q

how does the “Support Media” affect mobility

A

-support media should not bind the molecules being separated - has to be inert

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14
Q

What is agarose gel like as a support media

A

-inert
-isolated from agar
-most widely used in lab
-neutral
-doesnt bind protein, therefore doesnt affect migration
-electroendosmosis reduced
-needs small amounts of sample
-dried gel can be stored indefinitely

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15
Q

What is polyacrylamide gel like as a support media

A

-separates based on charge, size (due to gel pores) and weight
-layers of gel with different pore sizes
-inert

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16
Q

What is Cellulose acetate
like as a support media

A

-inert
-dry brittle film with 80% air
-when soaked in buffer, air is filled with electrolyte and film becomes pliable
-can be stored for a long time
-transparent after staining

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17
Q

how does the “time” affect mobility

A

-length of run in important
-the longer a run the better results are produced (good resolution)
-however longer runs can also cause increased diffusion of zones due to heat production

use minimal time to produce best seperation

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18
Q

how does the “Electrophoresis Buffers” affect mobility

A

-buffers have ions to help keep pH constant and carry applied current
-determine electrical charge on molecule, magnitude of charge and direction of migration

-barbital buffers (proteins), EDTA buffers (nucleic acids)

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19
Q

how does the “Ionic Strength of Buffers” affect mobility

A

-refers to charge and concentration of ions
-determines thickness of ion cloud around a molecule
-determines rate of migration
-determines sharpness or resolution of electrophortic zones

-decrease ionic strength gives increased mobility but poor buffering capability

-increase ionic strength gives decreased/slow mobility, sharper zones and more heat

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20
Q

how does the “Effect of Increased Ionic Strength” affect mobility

A

-greater the ionic strength the greater the resistance to movement (decreased migration)
-cleaner fractions increased resolution

-increased heat production can denature heat labile proteins

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21
Q

how does the “Electroendosmosis” affect mobility

A
  • overall solvent movement
    -movement of buffer ions and buffer solvent relative to fixed support medium
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22
Q

why would you order SPE

A

-Unexplained weakness, fatigue, anemia, renal insufficiency, increased ESR
-Bence Jones proteinuria, heavy proteinuria in >40 year old patients
-Hypercalcemia
-Hyper gammaglobulinemia, -immunoglobulin deficiency
-Recurrent infections

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23
Q

What is SPE

A

IEP of albumin is 4.8
IEP of gamma globulin is 7.4

-use barbital buffer of pH 8.6 because at this pH all serum proteins are negatively charged and migrate toward the anode

-this buffer pH is farthest from IEP of albumin so it has the largest charge and moves the fastest

-gamma globulins have smallest net charge and move the smallest amount

-reference sample and PT samples are applied to the cathode end of gel

24
Q

how to fix and stain after electrophoresis

A

fix - immobilize the bands,
dilute acids acetic acid, trochloroacetic acid and acid alcohol

stain using
-Amido Black, Ponceau S, Coomassie Brilliant Blue
-lipoproteins (Oil Red O, Sudan Black B), or CSF proteins (silver nitrate).
-type of stain varies with what is being separated

Chemiluminescence for igE using Anti IgE labelled with enzyme _dioxetant P

25
Q

Quantitation in PE

A

-use a densitometer to quantitate fractions
-% of total protein or absolute concentration (g/L)
-measures reflectance or transmittance
-the denser the stain on the band the more light it absorbs
signal for each band is seen as peak, area under the peak are calculated and % is converted into concentration

26
Q

Densitometric scan: Serum Proteins what are the peaks seen as

A

5 peak = 5 stained zones
-usually only 5 bands are seen on healthy people on an electrophoretogram. Proteins in bands can be quantitated separately by immunoassay
-in diseased states bands can increase or decrease

-peaks are homogenous with one protein or hetero with several
-homogenous proteins like albumin give narrow band
-width of fraction depends on number of proteins

27
Q

What is albumin

A

-most abundant serum protein
-elevated only when dehydrated
-decreases are of clinical interest - chronic liver disease, nephrotic syndrome, extensive burns, malabsorption can be non specific due to inflammatory reactions

28
Q

What is alpha 1 globulin

A

-alpha-1-antitrypsin, alpha-1-acid glycoprotein, and alpha-1-lipoprotein
-increases indicate active tissue damage like inflammation, malignancy and post op conditions

29
Q

What is alpha 2 globulin

A

-consists of 3 bands alpha-2 macroglobulin (most anodal), haptoglobin, and beta-lipoprotein (most cathodal and can also run in the beta zone)
-a raised fraction with a reduction of others can be nephrotic syndrome , protein losing issues
-haptoglobin is variable genetically and can migrate in the same broad band as beta 2 macroglobulin instead of alone

30
Q

Nephrotic syndrome

A

decrease in albumin
decrease in gamma globulins
increase in alpha 2 globulins

31
Q

What is beta globulin

A

made up of
transferrin
beta lipoprotein
complement C3 (heat labile and decreases if stored at room temp)
-visible changes here are uncommon

32
Q

what gamma globulins

A

consists of IgA, IgM, IgG

raised gamma globulins in
Chronic infections
cirrhosis of liver
autoimmune disorder

decreased in
Nephrotic syndrome (increased loss)
Malnutrition or immun0- deficiency (decreased synthesis)
Secondary suppression of synthesis

33
Q

What will you have if you have an increase in a single globulin out of the three

A

Multiple myeloma
Waldenstrom’s macroglobulinemia
Bench-Jones proteinemia

34
Q

What will be seen on monoclonal gammopathies

A

-Disorder of Ig synthesis due to b cell clone syntesis
-increase in plasma cells with single spike in M protein in beta gamma region
-if there is M protein there is a decrease in normal Ig
-if there is high M protein and decreased levels of Ig and can be associated with a malignant clinical course
-biclonal with increase in Beta and gamma and alb

seen as increase in peak at gamma region because of multiple myeloma

35
Q

what is seen in cirrhosis of liver

A

decrease of albumin
increase in gamma
bridging between beta and gamma

36
Q

what will you see in polyclonal gammopathy

A

=hypergammaglobulinemia
-increase of all gamma globulins
-decreased albumin
due to chronic infection
liver disease
burn
autoimmune disease

37
Q

what will you see in liver disease

A

decreased albumin
increased gamma globulins (polyclonal)
beta gamma bridging

38
Q

what is hypogammaglobulinemia

A

decreased or absent gamma globulin due to congitenal disorder (IgA or IgG def) and immunosuppressive therapy

39
Q

what will you see in viral hepatitis

A

decrease in albumin
increase in gamma globulins

40
Q

What will you see in chronic inflammation

A

increase in APRs - AAT, AAC, HAP, CER, C3/4 CRP
-non specific and general reaction to inflammation
-each APR rises at different rates
-seen with decrease in albumin synthesis so your TP will change

41
Q

how can you apply electrophoresis
spinal fluid proteins

A

-agarose gel electrophoresis
-shows albumin as major band as it makes up most of the CSF protein
-there will be a extra pre albumin fraction

-pre alb, alb, antityrp, transferrin, csf specific transferrin
-on abnormal patient you will see OLIG bands

42
Q

What are OLIG banding in spinal fluid proteins

A

-seen in gamma fraction in pt with MS
-can be seen very early and will be in the bottom of gel toward you

43
Q

how can you apply electrophoresis
isoenzymes

A

, LD, ALP, CK
-different types of LD banding indicates which organ is damaged
LD 1 - LD 5

increase in1,2, 3, 5 for MI
Increase in 1, 2, and 3 for pernicious or hemolytic anemia
increase in 4 and 5 = active liver disease
increase in all = malignant tumor shock

Alp comes from the liver, bone and intestine there is one band where liver and bone ALP overlaps

44
Q

how can you apply electrophoresis
isoenzymes CK

A

found in cardiac, brain and skeletal muscles
-if there is heart and brain damage you will see 3 distinct electrophoretic bands

CKMM increase Excessive Muscular activity
CKMM and ckmb increase - MI
CKMM and a tiny CKMB = duchennes muscular dystrophy

45
Q

how can you apply electrophoresis
Lipoproteins

A

-lipids are transported in plasma
-chylomicrons transport dietary lipids which will not be seen in fasting patient made up of :
-pre beta transports endogenous lipids
-B transports cholesterol
-Alpha is made up of protein bound phospholipids

lipoproteins can be separated into 5 types 1-5

46
Q

What to watch for in electrophoresis

A

stain solution
-replace if they are leaching or if there is staining
-stains should be stored in tight containers to avoid evaporation

Unequal migration rates
-dirty electrodes which support drying out

use small samples to avoid overloading with albumin because it is 10x more concentrated than alpha 1 and we need to be able to detect this as well

47
Q

What to watch for in electrophoresis
band irregularities

A

-distortion because of bent applications, bubbles, a wet or dry support
-artefacts, hemolysed samples, fibrinogen, split albumin or a widened albumin zone can cause unusual bands
-denatured proteins can give atypical band

make sure you use controls so you dont mistake these for reportable band

48
Q

What to watch for in electrophoresis
buffers

A

-buffers can be good culture media so keep them refridgerated because cold buffers improve resolution and minimized evaporation
-throw out small buffer volumes because they can cause pH changes due to electrolysis of water during electrolysis
-large volumes will not be as affected

49
Q

What will hemolysed samples be seen as

A

-increase in b2 area where the hemoglobin migrates or a band between a2 and b2 from the Hb-HAP complex

50
Q

What will fibrinogen in samples be seen as

A

show near application point which is why we use serum samples to avoid this extra band

51
Q

why will you see a spilt albumin peak or a wide peak

A

wide - drug that is albumin bound

split - benign condition called bisalbuminemia

52
Q

What will you have to do before putting the sample in the gel

A

blot twice ; once after removing from package and next to remove excess sample
-make sure you leave time for samples to diffuse into gel before blotting

let dry before you prep for staining

53
Q

What will you see in Hb electrophoresis

A

hbA with small HbF and HbA2
the HbA will migrate to beta region

54
Q

What is HBA1C

A

slow and irreversible
assesses degree of glycemic control in a diabetic patient
-when glucose reacts with amino group in Hb it implies high blood glucose and poor control

A1c depends on RBC lifespan and glucose levels in blood
-youll be able to see glycemic levels over the past 6-8 weeks

55
Q

how will heat affect gets

A

evaporates solvent from the ends of support medium
-drying will cause “wick up” on both ends of support to replace moisture loss

Clinical Chemistry (8 decks)

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